WO2017176963A1 - Production acellulaire d'acide ribonucléique - Google Patents

Production acellulaire d'acide ribonucléique Download PDF

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Publication number
WO2017176963A1
WO2017176963A1 PCT/US2017/026285 US2017026285W WO2017176963A1 WO 2017176963 A1 WO2017176963 A1 WO 2017176963A1 US 2017026285 W US2017026285 W US 2017026285W WO 2017176963 A1 WO2017176963 A1 WO 2017176963A1
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Prior art keywords
rna
cell lysate
thermostable
kinase
kinases
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PCT/US2017/026285
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English (en)
Inventor
William Jeremy BLAKE
Drew S. CUNNINGHAM
Daniel Maceachran
James Robbins ABSHIRE
Mehak GUPTA
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Greenlight Biosciences, Inc.
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Priority to MYPI2018703665A priority Critical patent/MY195729A/en
Priority to KR1020187032149A priority patent/KR102536687B1/ko
Priority to RU2018138975A priority patent/RU2018138975A/ru
Priority to CA3020312A priority patent/CA3020312A1/fr
Priority to CN201780033464.4A priority patent/CN109196109A/zh
Priority to SG11201808721YA priority patent/SG11201808721YA/en
Priority to EP17722192.6A priority patent/EP3440215A1/fr
Priority to JP2018553092A priority patent/JP7011599B2/ja
Application filed by Greenlight Biosciences, Inc. filed Critical Greenlight Biosciences, Inc.
Priority to BR112018070506A priority patent/BR112018070506A2/pt
Priority to CR20180525A priority patent/CR20180525A/es
Priority to KR1020237017261A priority patent/KR20230079463A/ko
Priority to AU2017246458A priority patent/AU2017246458B2/en
Priority to EP23190390.7A priority patent/EP4293104A3/fr
Publication of WO2017176963A1 publication Critical patent/WO2017176963A1/fr
Priority to IL262138A priority patent/IL262138A/en
Priority to JP2022004117A priority patent/JP2022062056A/ja
Priority to AU2022268349A priority patent/AU2022268349A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04001—Polyphosphate kinase (2.7.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04006—Nucleoside-diphosphate kinase (2.7.4.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/13—Exoribonucleases producing 5'-phosphomonoesters (3.1.13)
    • C12Y301/13001—Exoribonuclease II (3.1.13.1)

Definitions

  • RNA Ribonucleic acid
  • RNA acts as the key messenger of information in cells, carrying the instructions from DNA for the regulation and synthesis of proteins.
  • RNA is of interest in biotechnology as synthetically modulating mRNA levels in cells (positively through the introduction of mRNA or negatively through the introduction of siRNA or dsRNA) has applications in fields such as agricultural crop protection, anti-cancer therapeutics, and vaccines.
  • RNA interference refers to a cellular mechanism that uses the DNA sequence of a gene to turn the gene "off - a process referred to as "silencing.”
  • dsRNA double-stranded RNA
  • Functional single-stranded (e.g. mRNA) and double-stranded RNA molecules have been produced in living cells and in vitro using purified, recombinant enzymes and purified nucleotide triphosphates (see, e.g., European Patent No. 1631675 and U.S. Patent Application Publication No. 2014/0271559 Al, each of which is incorporated herein by reference). Nonetheless, the production of RNA at scales enabling widespread commercial application is currently cost-prohibitive.
  • RNA RNA from a biomass material
  • a series of kinases a series of kinases
  • a corresponding nuclei acid e.g., DNA
  • the methods, compositions, cells, constructs, and systems of the present disclosure are used for the production of RNA under cell-free conditions, for example, using at least one cell lysate, a combination of purified proteins, or a combination of cell lysate(s) and purified protein(s).
  • the present disclosure is based, in some embodiments, on the conversion of RNA from biomass (e.g., endogenous cellular RNA) to desired synthetic RNA (e.g., synthetic single-stranded or double-stranded RNA) using a cell lysate.
  • RNA from biomass e.g., endogenous RNA
  • RNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • NMPs 5'-nucleoside monophosphates
  • these nucleases are inactivated or partially inactivated (e.g., via heat inactivation), and the NMPs are phosphorylated to ribonucleotide triphosphates (NTPs) by a series of thermostable kinase activities ( Figure 1, reaction 2).
  • NTPs ribonucleotide triphosphates
  • Figure 1, reaction 3 thermostable kinase activities
  • the desired synthetic RNA optionally may be purified from the cell lysate.
  • RNA ribonucleic acid
  • the methods comprising: (a) incubating at least one cell lysate mixture that comprises (i) RNA and (ii) at least one enzymatic activity selected from the group consisting of enzymatic activities that depolymerize RNA, thermostable kinase activities, and thermostable RNA polymerase activities, under conditions that result in depolymerization of RNA to produce a cell lysate mixture that comprises nucleoside monophosphates; (b) heating the cell lysate mixture produced in step (a) to a temperature (e.g., 50-80 °C) that inactivates or partially inactivates endogenous nucleases and phosphatases without completely inactivating the thermostable kinase activities and thermostable RNA polymerase activities, to produce a cell lysate mixture that comprises heat-inactivated nuclea
  • a temperature e.g., 50-80 °C
  • the cell lysate mixture may comprise a single cell lysate obtained from cells that comprise RNA and express at least one enzyme (including at least one fusion enzyme) that acts as a ribonuclease, acts as a kinase, and/or acts as a RNA polymerase.
  • at least one enzyme including at least one fusion enzyme
  • the cell lysate mixture may comprise at least two (e.g., at least 3, 4, 5, or 6) cell lysates, wherein at least one cell lysate is obtained from cells that comprise RNA, and at least one cell lysate (e.g., at least 2, 3, 4, or 5) is obtained from cells that express at least one enzyme that acts as a nuclease, acts as a kinase, and/or acts as a RNA polymerase.
  • An enzyme or fusion enzyme is considered to "act as a nuclease” if the enzyme of fusion enzyme exhibits nuclease activity (cleaves or depolymerizes a nucleic acid; e.g., RNase R).
  • An enzyme or fusion enzyme is considered to "act as a kinase” if the enzyme of fusion enzyme exhibits kinase activity (catalyzes the transfer of a phosphate group from one molecule to another molecule; e.g. polyphosphate kinase).
  • An enzyme or fusion enzyme is considered to "act as a polymerase” if the enzyme of fusion enzyme exhibits polymerase activity (assembles nucleotides to produce nucleic acids; e.g., RNA polymerase).
  • the RNA of step (a) is messenger RNA (mRNA), transfer RNA (tRNA), or ribosomal RNA (rRNA).
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • the cell lysate mixture comprises at least one ribonuclease, at least one thermostable kinase, and/or at least one RNA polymerase (e.g., a thermostable RNA polymerase).
  • a thermostable RNA polymerase e.g., a thermostable RNA polymerase
  • the use of fusion enzymes is also encompassed by the present disclosure.
  • the cell lysate mixture may comprise a fusion of a ribonuclease and a kinase, or a fusion of multiple kinases.
  • Other fusion enzymes are encompassed by the present disclosure.
  • engineered cells, cell lysates, and cell lysate mixtures comprising at least one nucleoside monophosphate kinase (e.g., thermostable nucleoside monophosphate kinase), at least one nucleoside diphosphate kinase (e.g., thermostable nucleoside diphosphate kinase), and at least one polyphosphate kinase (e.g., thermostable polyphosphate kinase).
  • the cells may also comprise at least one ribonuclease and/or at least one RNA polymerase (e.g., thermostable RNA polymerase).
  • methods of producing (biosynthesizing) RNA comprise (a) lysing cultured cells (e.g., engineered cells) that comprise RNA (e.g., mRNA, tRNA, and/or rRNA), RNase R, thermostable kinases (e.g., PfPyrH, TthAdk, TthCmk, PfGmk, AaNdk, TePpk, and/or PPK2 (e.g., see Table 6), and a thermostable T7 RNA polymerase, thereby producing a cell lysate, (b) incubating the cell lysate produced in step (a) under conditions that result in depolymerization of RNA to 5'-NMPs, thereby producing a cell lysate that comprises 5'-NMPs, (c) heating the cell lysate produced in step (b) to 60-80 °C to inactivate endogenous nucleases
  • RNA e.g
  • RNA In some embodiments, the RNA, RNase R, thermostable kinases, and
  • thermostable T7 RNA polymerase are contained in a single strain of cultured cells (e.g., engineered cells).
  • cultured cells e.g., engineered cells
  • containing a subset of the above activities/components are lysed, and the lysates combined to generate a cell lysate mixture that comprises all the enzymatic activities described in step (a) above.
  • enzymatic activities in the form of purified enzymes, are added to the lysates described in step (a) above.
  • lysates and/or purified proteins are combined before the heat-inactivation step described in step (c) above.
  • lysates and/or purified proteins are combined after the heat inactivation step described in step (c) above.
  • the RNA of interest may be any form of RNA, including single-stranded RNA and double-stranded RNA.
  • the RNA of interest may be messenger RNA (mRNA), anti sense RNA, micro RNA, small interfering RNA (siRNA), or a short hairpin RNA (shRNA).
  • mRNA messenger RNA
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • RNAi RNA interference
  • Figure 1 shows a schematic of cell-free RNA production as described herein.
  • Cells containing RNA from biomass e.g., endogenous RNA
  • a nuclease, a thermostable kinase, and/or a thermostable RNA polymerase are lysed (or combined and lysed), and the resulting cell lysate(s) is/are incubated under conditions that result in depolymerization of the RNA.
  • the cell lysate is then heated to inactivate the nuclease and any endogenous phosphatases (without inactivating the thermostable kinase and thermostable RNA polymerase).
  • RNA of interest e.g., ssRNA or dsRNA
  • individual, purified pathway enzymes e.g., RNA polymerase, such as thermostable RNA polymerase
  • RNA polymerase such as thermostable RNA polymerase
  • the engineered cells used to produce the cell lysate do not express one or more of the enzymatic activities described above, e.g., a nuclease, a thermostable kinase and/or a thermostable RNA polymerase.
  • Figure 2A shows a schematic of a polyphosphate-dependent kinase pathway for energy generation.
  • Figure 2B shows a schematic of additional exemplary energy conversion pathways for use in the methods and systems of the present disclosure.
  • a UMP kinase e.g., obtained from Pyrococcus furiosus
  • a polyphosphate kinase e.g., obtained from
  • Meiothermus ruber Meiothermus silvanus, Deinococcus geothermalis, Anaerolinea thermophila, , Chlorobaculum tepidum, Oceanithermus profundus, Roseiflexus castenholzii, Roseiflexus sp., or Truepera radiovctrix
  • NDP kinases e.g., encoded by an Aquifex aeolicus ndk gene
  • a polyphosphate kinase may be used to convert UDP to UTP.
  • a CMP kinase (e.g., obtained from Thermus thermophilus) and a polyphosphate kinase may be used to convert CMP to CDP, and NDP kinases and a polyphosphate kinase may be used to convert CDP to CTP.
  • a GMP kinase (e.g., obtained from Thermotoga maritimd) and a polyphosphate kinase may be used to convert GMP to GDP, and NDP kinases and a polyphosphate kinase may be used to convert GDP to GTP.
  • An AMP kinase e.g., obtained from Thermus thermophilus
  • a polyphosphate kinase may be used to convert AMP to ADP
  • NDP kinases e.g., encoded by an Aquifex aeolicus ndk gene
  • a polyphosphate kinase may be used to convert ADP to ATP
  • a Class III PPK2 enzyme see, e.g., Table 6) may be used to convert AMP to ATP.
  • Figure 3A shows a schematic of an example of a DNA template used for the biosynthesis of double-stranded RNA.
  • the DNA template encoded as part of a plasmid, contains a single coding region including of a promoter operably linked to the coding region of interest and one or more terminators. Following transcription, the RNA folds into a hairpin structure through intramolecular nucleotide base pairing.
  • the DNA template either alone or encoded as part of a plasmid, contains two complementary domains (1 and 3), separated by domain 2.
  • Figure 3B shows a schematic of another example of a DNA template used for biosynthesis of double-stranded RNA.
  • the DNA template contains converging promoter sequences on complementary strands.
  • FIG. 3C shows a schematic of another example of a DNA template used for biosynthesis of double-stranded RNA.
  • the DNA template, encoded as part of a plasmid contains converging promoter sequences operably linked to coding regions of interest on complementary strands, as well as one or more terminator sequences to prevent read-through transcription.
  • Figure 3D shows a schematic of another example of a DNA template used for biosynthesis of double-stranded RNA.
  • the DNA template, encoded as part of a plasmid contains independent cassettes, each including of a promoter operably linked to the coding region of interest and one or more terminators, driving transcription of
  • FIG. 3E shows a schematic of another example of a DNA template used for biosynthesis of double-stranded RNA.
  • DNA-dependent RNA polymerase is used to produce ssRNA template, and the RNA-dependent RNA polymerase is used to produce double-stranded RNA.
  • FIG. 4 shows a schematic of another example of a cell-free RNA production method of the present disclosure.
  • the process starts with a single fermentation vessel in which engineered cells are produced using standard fermentation techniques. Biomass generated from the fermentation is optionally concentrated by microfiltration (MF) followed by lysis via mechanical homogenization, for example. The lysate is then pumped into a second fermentation vessel wherein the expressed nuclease enzymes convert RNA to its monomeric constituents.
  • MF microfiltration
  • the entire reaction is heated to inactivate any endogenous phosphatase or nuclease ⁇ e.g., RNase) activities as well as any other exogenous/introduced cellular ⁇ e.g., nuclease) activity that would be detrimental to RNA product stability and/or fidelity.
  • polyphosphate is fed to the reaction as a source of high- energy phosphate for the phosphorylation of NMPs to NTPs via a series of thermostable kinases, followed by polymerization to dsRNA.
  • Downstream processing may be used to increase purity to as much as 99% (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%) dsRNA by weight.
  • processing may be used to increase purity to 50-60%), 50- 70%, 50-80%, 50-90%, 50-95%, 70-80%, 70-90%, or 70-95%.
  • An examplary downstream process starts with the addition of a protein precipitating agent ⁇ e.g., ammonium acetate) followed by removal of protein, lipids and some DNA from the product stream by disc stack centrifugation (DSC) or tangential flow filtration (TFF). Ultrafiltration is then implemented to remove salts and reduce volume. Addition of lithium chloride to the product stream leads to precipitation of the dsRNA product, is subsequently be separated from the bulk liquid using disc stack centrifugation, yielding an 80% purity dsRNA product stream.
  • a protein precipitating agent ⁇ e.g., ammonium acetate
  • DSC disc stack centrifugation
  • THF tangential flow filtration
  • Figures 5A-5B show a comparison of ribonuclease activities by digestion of purified E. coli RNA.
  • Figure 5A Release of acid-soluble nucleotides (mononucleotides and short oligonucleotides) with nuclease treatment was most rapid with Benzonase, RNase A, RNase R, and Nuclease PI .
  • Figure 5B LC-MS analysis of reaction products demonstrated NMP release from RNA with RNase R and Nuclease PI treatment.
  • FIG. 6 is a graph showing depolymerization of lysate RNA using exogenous RNase R, with products analyzed by UPLC to specifically identify 5'-NMPs.
  • lysates exhibited endogenous RNase activity that led to the slow accumulation of 5'-NMPs (solid dark gray line).
  • Addition of exogenous RNase R led to rapid 5'-NMP release (dashed dark gray line) without affecting rates of 2' or 3' NMP accumulation (light gray lines).
  • overexpression of RNase R accelerates the rate of polymeric RNA to 5'- NMP conversion, reducing the deleterious effects of phosphatase/nuclease activities present in the extract. Experiments were performed at a final concentration of 50% lysate.
  • Figures 7A-7C show results of RNase R overexpression in 1L bioreactors cultures grown in batch phase.
  • Figure 7A SDS-PAGE analysis of protein expression in duplicate cultures.
  • Empty vector cultures contained an empty protein expression vector (pETDuet-1).
  • RNase R cultures contained E. coli rnr cloned into pETDuet-1.
  • Samples from induced cultures (+) exhibited strong expression of RNase R (MW 92.9 kDa with C-terminal hexahistidine tag) indicated by arrow at right.
  • ⁇ Figure 7B Growth kinetics (encompassing pre- and post-induction growth) of Empty Vector (dark gray) and RNase R-expressing strains (light gray) demonstrated that RNase R overexpression was not deleterious to cell growth. Dashed lines represent exponential curve fits.
  • Figure 7C Overexpressed RNase R was active in lysates of batch-grown biomass, releasing acid-soluble nucleotides. In the empty vector strain (solid dark gray line), adding exogenous RNase R increased the rate of nucleotide release (dashed dark gray line). In contrast, strains expressing RNase R exhibited rapid nucleotide release upon lysis (solid light gray line). Adding exogenous RNase R did not increase rates of nucleotide release or final nucleotide yields (dashed light gray line).
  • Figure 8 is a graph describing the effects of chelating Mg 2+ on depolymerization rates in high-density lysates.
  • Lysates prepared from biomass containing Empty Vector (dark gray) were insensitive to EDTA.
  • Lysates with overexpressed RNase R (light gray) exhibited rapid RNA depolymerization with Mg 2+ removal, with 8 mM EDTA providing maximum depolymerization rates.
  • Experiments were performed at a final concentration of 90% lysate.
  • Figures 9A-9D show graphs demonstrating stability of exogenous isotopically- labeled "heavy" MPs (h MPs) in lysates:
  • Figure 9 A hAMP was relatively stable in lysates, with 90% remaining after a 1 hour incubation at 37 °C.
  • Figure 9B hCMP was degraded in lysates, with 70% remaining after approximately 30 minutes. Addition of 10 mM sodium orthovanadate (dotted line) (an inhibitor of several phosphatases and kinases) significantly improved stability.
  • Figure 9C hUMP was degraded in lysates, with 70% remaining after approximately 20 minutes. Sodium phosphate (150 mM) (dashed line) and sodium orthovanadate (dotted line) significantly improved stability.
  • Figure 9D hGMP was degraded in lysates, with 70% remaining after approximately 10 minutes.
  • orthovanadate significantly improved stability, with 70% hGMP remaining after 30 minutes.
  • Figures 11A-11B are graphs demonstrating the effect of heat inactivation on the stability of NMPs and dsRNA in lysates.
  • Figure HE Heat inactivation stabilized reactants and products of transcription reactions in lysates.
  • Lysates were pre-incubated for 15 minutes at the indicated temperature, then the temperature was lowered to 37 °C and transcription reactants were added. Heat inactivation at 70 °C and 80 °C, but not 60 °C stabilized substrates and products sufficiently to produce a detectable transcription product similar to the positive control (no lysate).
  • Figure 12 is a graph demonstrating temperature-dependent activity of UMP kinase from P. furiosus (PfPyrH), quantified by luciferase assay for ATP consumption.
  • the specific activity of purified PfPyrH was largely insensitive to incubation temperature.
  • FIG 13 is a graph demonstrating temperature-dependent activity of AMP kinase from T. thermophilus (TthAdk) compared to Adk from E. coli (EcAdk), measured via luciferase. Purified EcAdk was active at temperatures below 60 °C. TthAdk had higher specific activity, with a maximum at 70 °C.
  • Figure 14 is a graph demonstrating temperature-dependent activity of CMP kinase from T. thermophilics (TthCmk), measured via luciferase. Purified TthCmk was relatively insensitive to temperature, with high activity from 37-80°C.
  • Figure 15 is a graph demonstrating temperature-dependent activity of GMP kinases from E. coli (EcGmk), T. thermophilus (TthGmk), and T. maritima (TmGmk), measured via luciferase.
  • EcGmk E. coli
  • TthGmk T. thermophilus
  • TmGmk T. maritima
  • Figure 16 is a graph of data demonstrating activity of purified DP kinase from A. aeolicus (AaNdk), measured via luciferase. Purified AaNdk was highly active from 37- 80°C, using ATP and GDP as substrates, with optimal activity at 50°C.
  • Figure 77 is a graph demonstrating activity of purified polyphosphate kinase 1 (PPK1) enzymes from . coli (EcPpk), Thermosynechococcus elongatus (TePpk), and Thermus thermophilus (TthPpk) measured via luciferase.
  • EcPpk was most active at temperatures ⁇ 60°C, while TePpk was optimally active at 70°C.
  • TthPpk exhibited relatively low activity.
  • Figure 18 is a graph demonstrating activity of commercially-available T7 RNA polymerases in buffer using conditions recommended by their respective manufacturers at 37°C. ThermoT7 and MegaScript polymerases exhibited higher specific activity than the NEB polymerase under the tested conditions with duplex DNA template ⁇ e.g. in Figure 3B).
  • Figure 19 is a graph comparing T7 RNA polymerase activities in dilute lysates at 37°C and 50°C under standardized reaction conditions with duplex DNA template. At 37°C, ThermoT7 exhibited the highest specific activity. At 50°C, only ThermoT7 had detectable activity, producing over 10 g/L/hr dsRNA.
  • Figure 20 is a graph demonstrating activity of ThermoT7 activities in buffer and high-density heat-inactivated lysates. ThermoT7 activity was highest in lysates clarified by centrifugation after heat-inactivation. Omitting the clarification step led to a 60% decrease in activity, although polymerase activity in unclarified matrix was greater than in buffer alone.
  • Figure 21 is a graph demonstrating tolerance of ThermoT7 to elevated
  • FIG 22A is an image of an SDS-PAGE gel showing expression and solubility data for A. thermophila PPK2 in E. coli strain GL16-170. MW: Unstained Protein Standard, Broad Range (New England Biolabs Cat # P7704). -: Pre-induction culture. +: Induced culture at harvest. L: Soluble protein in clarified lysates. A. thermophila PPK2: 33 kDa.
  • Figure 22B is a graph showing ATP production of A. thermophila PPK2 in heat-inactivated lysates. Closed circles denote ATP production from ADP. Open circles denote ATP production from AMP. For both substrates, A. thermophila PPK2 produces ATP at rates exceeding 400 mM/hr.
  • Figure 23 is an image of an agarose gel demonstrating application of thermostable Class III PPK2 for energy generation in cell-free dsRNA production.
  • Left lanes contain positive controls, demonstrating dsRNA synthesis from NTPs.
  • Middle lanes contain positive controls, demonstrating dsRNA synthesis from NMPs in nucleotide kinase-expressing lysates using exogenous ATP as an energy source.
  • Right lanes contain reactions demonstrating dsRNA synthesis from NMPs and HMP using nucleotide kinase and C. aerophila Ppk- expressing lysates.
  • cell-free RNA synthesis reactions are Mn 2+ independent. Reactions without polymerase are included as negative controls, illustrating background nucleic acid content of each lysate-containing reaction.
  • Figure 24 is an image of an agarose gel demonstrating application of thermostable Class III PPK2 for energy generation in cell-free dsRNA production.
  • Left lanes contain positive controls, demonstrating dsRNA synthesis from NTPs.
  • Middle lanes contain positive controls, demonstrating dsRNA synthesis from NMPs in nucleotide kinase-expressing lysates using exogenous ATP as an energy source.
  • Right lanes contain reactions demonstrating dsRNA synthesis from NMPs and HMP using nucleotide kinase and C. aerophila Ppk- expressing lysates. With C. aerophila PPK2, dsRNA synthesis proceeds in the absence of AMP kinase and exogenous ADP.
  • RNA or DNA nucleic acid
  • a single type of organism e.g., a population of bacterial cells
  • the engineered cells are grown (cultured) under conditions that result in enzyme expression.
  • the engineered cells may be grown to a desired cell density, and then expression of certain enzymes may be induced (activated).
  • transcription of certain enzymes may be under the control of an inducible promoter.
  • the cells e.g., engineered and/or non-engineered cells
  • lysed e.g., mechanically, chemically, or enzymatically disrupted
  • cells containing polymeric RNA e.g., mRNA, tRNA, and/or rRNA
  • mRNA, tRNA, and/or rRNA are mixed with the engineered cells containing pathway enzymes prior to the cell lysis step.
  • cell lysate(s) obtained from cells containing polymeric RNA is combined (mixed) with cell lysate(s) obtained from engineered cells containing pathway enzymes.
  • one or more purified pathway enzymes are combined (mixed) with cell lysate(s) obtained from engineered cells.
  • "Pathway enzymes” are enzymes required to biosynthesize the RNA of interest (e.g., starting from polymeric RNA).
  • the cell lysate (or cell lysate mixture) is incubated under conditions that result in nuclease-mediated (e.g., RNase-mediated) depolymerization of the host-derived (endogenous) RNA to a desired yield of 5'-nucleoside monophoshates (NMPs, or nucleoside monophosphates).
  • NMPs 5'-nucleoside monophoshates
  • the cell lysate (or cell lysate mixture) is then heated, in some embodiments, to inactivate the majority of host-derived enzymes, including
  • phosphatases and nucleases e.g., RNases
  • nucleases e.g., RNases
  • the cell lysate is incubated under conditions that result in phosphorylation of the NMPs to NTPs (nucleoside triphosphates) by thermostable kinases (e.g., thermostable nucleoside monophosphate kinases and nucleoside diphosphate kinases) using, for example, thermostable polyphosphate kinase and the addition of polyphosphate as the energy source.
  • thermostable kinases e.g., thermostable nucleoside monophosphate kinases and nucleoside diphosphate kinases
  • the resulting NTPs are subsequently polymerized to RNA by a RNA polymerase (e.g., thermostable RNA polymerase) using an engineered template (e.g., DNA template) present in the lysates (e.g., either expressed by the engineered cells and included as a cellular component of the cell lysate, or later added to the cell lysate).
  • a RNA polymerase e.g., thermostable RNA polymerase
  • an engineered template e.g., DNA template
  • Cell-free production is the use of biological processes for the synthesis of a biomolecule or chemical compound without using living cells.
  • the cells are lysed and unpurified (crude) portions or partially-purified portions, both containing enzymes, are used for the production of a desired product.
  • Purified enzymes may be added to cell lysates, in some embodiments.
  • cells are cultured, harvested, and lysed by high-pressure homogenization or other cell lysis method (e.g., chemical cell lysis).
  • the cell-free reaction may be conducted in a batch or fed-batch mode. In some instances, the enzymatic pathways fill the working volume of the reactor and may be more dilute than the intracellular environment.
  • cellular catalysts including catalysts that are membrane associated.
  • the inner membrane is fragmented during cell lysis, and the fragments of these membranes may form membrane vesicles. See, e.g., Swartz, AIChE Journal, 2012, 58(1), 5-13, incorporated herein by reference.
  • Cell-free methods, compositions, and systems of the present disclosure utilize cell lysates (e.g., crude or partially purified cell lysates), discussed in greater detail herein.
  • Cell lysates prepared, for example, by mechanical means (e.g., shearing or crushing), are distinct from chemically-permeabilized cells.
  • mechanical means e.g., shearing or crushing
  • inverted membrane vesicles are formed in the cell lysates.
  • Such inverted membrane vesicles are not produced through chemical cell permeabilization methods.
  • Cells that are lysed e.g., at least 75%, 80%, 85%), 90%, or 95%) are no longer intact.
  • permeabilized cells, which are intact cells containing perforations (small holes) are not considered lysed cells.
  • the methods provided herein are generally cell-free and use cell lysates, in some embodiments, it may be advantageous, at least for some steps of the methods, to use permeabilized cells. Thus, the present disclosure does not exclude the use of permeabilized cells in at least one step of the RNA production methods.
  • lysing cultured cells that comprise particular enzymes
  • the phrase is intended to encompass lysing a clonal population of cells obtained from a single culture (e.g., containing all the enzymes needed to synthesize RNA) as well as lysing more than one clonal population of cells, each obtained from different cell cultures (e.g., each containing one or more enzymes needed to synthesize RNA and/or the polymeric RNA substrate).
  • a population of cells (e.g., engineered cells) expressing one thermostable kinase may be cultured together and used to produce one cell lysate, and another population of cells (e.g., engineered cells) expressing a different thermostable kinase may be cultured together and used to produce another cell lysate.
  • two cell lysates, each comprising a different thermostable kinase may then be combined for use in a RNA biosynthesis method of the present disclosure.
  • RNA from biomass e.g., endogenous cellular RNA
  • desired synthetic RNA using a cell lysate through a cell-free process involving a series of enzymatic reactions.
  • RNA e.g., endogenous RNA
  • RNA from biomass e.g., endogenous RNA
  • RNA from biomass typically includes ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), other RNAs, or a combination thereof.
  • RNA of interest is ssRNA (e.g., mRNA). In some embodiments, the RNA of interest is dsRNA.
  • RNA e.g., endogenous RNA
  • the amount of RNA (e.g., endogenous RNA) required to synthesize a RNA of interest may vary, depending on, for example, the desired length and yield of the RNA of interest as well as the nucleotide composition of the RNA relative to the nucleotide composition of the RNA (e.g., endogenous RNA) of the cell (e.g., E. coli cell).
  • RNA (e.g., endogenous RNA) content ranges from 5-50% of the total cell mass.
  • the mass of the starting material can be calculated, for example, using the following equation: (kilogram (kg) of RN A/kilogram of dry cell weight) x 100%.
  • Endogenous RNA may be depolymerized or degraded into its constituent monomers by chemical or enzymatic means. Chemical hydrolysis of RNA, however, typically produces 2'- and 3'-NMPs, which cannot be polymerized into RNA. Thus, the methods, compositions, and systems as provided herein primarily use enzymes for the depolymerization of endogenous RNA.
  • An "enzyme that depolymerizes RNA” catalyzes the hydrolysis of the phosphodiester bonds between two nucleotides in a RNA.
  • an enzyme that depolymerizes RNA converts RNA (polymeric RNA) into its monomelic form— nucleoside monophosphates (NMPs).
  • enzymatic depolymerization of RNA may yield 3'-NMPs, 5'-NMPs or a combination of 3'-NMPs and 5'- NMPs. Because it is not possible to polymerize 3'-NTPs (converted from 3'-NDPs, which are converted from 3 '-NMPs), enzymes (e.g., RNase R) that yield 5 '-NMPs (which are then converted to 5'-NDPs, and then 5'-NTPs) are preferred. In some embodiments, enzymes that yield 3 '-NMPs are removed from the genomic DNA of the engineered cell to increase efficiency of RNA production. In some embodiments, the enzyme used for RNA
  • the concentration of RNase R used is 0.1-1.0 mg/mL (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9. or 1.0 mg/mL). In some embodiments, the concentration of RNase R used is 0.4-0.6 mg/mL. In some embodiments, the concentration of RNase R used is 0.5 mg/mL. In some embodiments, the concentration of RNase R used is greater than 1.0 mg/mL.
  • Examples of enzymes that depolymerize RNA include, without limitation, nucleases, including ribonucleases (RNases, e.g., RNase R) and phosphodiesterases.
  • Nucleases catalyze the degradation of nucleic acid into smaller components (e.g., monomers, also referred to as nucleoside monophosphates, or oligonucleotides). Phosphodiesterases catalyze degradation of phosphodiester bonds. These enzymes that depolymerize RNA may be encoded by full length genes or by gene fusions (e.g., DNA that includes at least two different genes (or fragments of genes) encoding at two different enzymatic activities).
  • RNase functions in cells to regulate RNA maturation and turn over.
  • Each RNase has a specific substrate preferences— dsRNA or ssRNA.
  • a combination of different RNases, or a combination of different nucleases generally, may be used to depolymerize biomass-derived polymeric RNA (e.g., endogenous RNA).
  • biomass-derived polymeric RNA e.g., endogenous RNA
  • 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 different nucleases may be used in combination to depolymerize RNA.
  • nucleases may be used in combination to depolymerize RNA.
  • nucleases for use as provided herein are included in Table 1.
  • the nuclease used is RNase R.
  • Enzymes that depolymerize RNA may be endogenous to a host cell (host-derived), or they may be encoded by engineered nucleic acids exogenously introduced into a host cell (e.g., on an episomal vector or integrated into the genome of the host cell).
  • engineered nucleic acids encoding enzymes that depolymerize RNA are operably linked to an inducible promoter.
  • nucleic acids may be engineered to encode enzymes (e.g., RNases) that are relocated to or are sequestered in the periplasm of a host cell so that activity of the enzyme does not interfere with cell growth or other metabolic processes.
  • enzymes e.g., RNases
  • the relocated enzyme is released from the periplasm, brought into contact with the endogenous RNA, and depolymerizes the RNA into monomeric form. See, e.g., International Publication No. WO 2011/140516, published November 10, 2011, incorporated herein by reference.
  • RNA "Conditions that result in depolymerization of RNA” are known in the art or may be determined by one of ordinary skill in the art, taking into consideration, for example, optimal conditions for nuclease (e.g., RNase) activity, including pH, temperature, length of time, and salt concentration of the cell lysate as well as any exogenous cofactors. Examples including those described previously (see, e.g., Wong, C.H. et al. J. Am. Chem. Soc, 105: 115-117, 1983), EP1587947B1, Cheng ZF, Irishr MP. J Biol Chem. 277:21624-21629, 2002).
  • nuclease e.g., RNase
  • metal ions e.g., Mg 2+
  • Mg 2+ metal ions
  • the concentration of metal ion (e.g., Mg 2+ ) is 8 mM or less (e.g., less than 8 mM, less than 7 mM, less than 6 mM, less than 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, less than 1 mM, less than 0.5 mM).
  • the concentration of metal ion (e.g., Mg 2+ ) is 0.1 mM-8 mM, 0.1 mM-7 mM, or 0.1 mM-5 mM.
  • the pH of a cell lysate during a RNA depolymerization reaction may have a value of 3.0 to 8.0.
  • the pH value of a cell lysate is 3.0-8.0, 4.0-8.0, 5.0-8.0, 6.0-8.0, 7.0-8.0, 3.0-7.0, 4.0-7.0, 5.0-7.0, 6.0-7.0, 3.0-6.0, 4.0-6.0, 5.0-6.0, 3.0-5.0, 3.0-4.0, or 4.0-5.0.
  • the pH value of a cell lysate is 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.
  • the pH value of a cell lysate is 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. The pH of a cell lysate may be adjusted, as needed.
  • the temperature of a cell lysate during a RNA depolymerization reaction may be 15 °C to70 °C. In some embodiments, the temperature of a cell lysate during a RNA depolymerization reaction is 15-60 °C, 15-50 °C, 15-40 °C, 15-30 °C, 25-70 °C, 25-60 °C, 25-50 °C, 25-40 °C, 30-70 °C, 30-60 °C, or 30-50 °C. In some embodiments, the
  • a cell lysate during a RNA depolymerization reaction may be incubated for 5 minutes (min) to 72 hours (hrs). In some embodiments, a cell lysate during a RNA
  • RNA depolymerization reaction is incubated for 5-10 min, 5-15 min, 5-20 min, 5-30 min, or 5 min- 48 hrs.
  • a cell lysate during a RNA depolymerization reaction may be incubated for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 1 1 hrs, 12 hrs, 18 hrs, 24 hrs, 30 hrs, 36 hrs, 42 hours, or 48 hours.
  • a cell lysate during a RNA depolymerization reaction is incubated for 24 hours at a temperature of 37 °C. In some embodiments, a cell lysate during a RNA depolymerization reaction is incubated for 5-10 min at a temperature of 37 °C. In some embodiments, a cell lysate during a RNA depolymerization reaction has a pH of 7.0 and is incubated for 15 minutes at a temperature of 37 °C. In some embodiments, a cell lysate during a RNA depolymerization reaction may be incubated under conditions that result in greater than 65% conversion of RNA to 5'-NMPs. In some embodiments, RNA is converted to 5'-NMPs at a rate of (or at least) 50 mM/hr, 100 mM/hr or 200 mM/hr.
  • salt is added to a cell lysate, for example, to prevent enzyme aggregation.
  • sodium chloride, potassium chloride, sodium acetate, potassium acetate, or a combination thereof may be added to a cell lysate.
  • the concentration of salt in a cell lysate during a RNA depolymerization reaction may be 5 mM to 1 M.
  • the concentration of salt in a cell lysate during a RNA depolymerization reaction 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 500 mM, 750 mM, or 1 M.
  • the cell lysate comprises a mixture that includes 40-60 mM potassium phosphate, 1-5 mM MnCl 2 , and/or 10-50 mM MgCl 2 (e.g., 20 mM MgCl 2 ).
  • buffer is added to a cell lysate, for example, to achieve a particular pH value and/or salt concentration.
  • buffers include, without limitation, phosphate buffer, Tris buffer, MOPS buffer, HEPES buffer, citrate buffer, acetate buffer, malate buffer, MES buffer, histidine buffer, PIPES buffer, bis-tris buffer, and ethanolamine buffer.
  • RNA results in the production of 5'-NMP, including 5'-AMP, 5'-UMP, 5'-CMP, and 5'-GMP. While the NMP may be present in the cell lysate at relatively equimolar amounts, depolymerization of RNA does not result in any predetermined ratio of NMPs.
  • 50-98% of the endogenous RNA in a cell upon lysis is converted to (depolymerized to) 5'-NMPs.
  • 50-95%, 50-90%, 50-85%, 50-80%, 75-98%, 75-95%, 75-90%, 75-85% or 75-80% RNA is converted to (depolymerized to) 5'- NMPs.
  • 65-70%) of the endogenous RNA in a cell upon lysis is converted to (depolymerized to) 5'-NMPs. Lower yields are also acceptable.
  • RNA from biomass ⁇ e.g., endogenous RNA
  • nucleases and phosphatases which may have deleterious effects on RNA biosynthesis.
  • Escherichia coli has numerous phosphatases, many of which dephosphorylate NTPs, NDPs, and NMPs. Dephosphorylation of NMPs following RNA depolymerization results in the accumulation of the non- phosphorylated nucleosides and a loss of usable NMP substrate, thus reducing synthetic RNA yield.
  • NMPs, NDPs, or NTPs dephosphorylation of NMPs, NDPs, or NTPs following RNA depolymerization results in futile energy cycles (energy cycles that produce a low yield of synthetic RNA) during which NMPs are phosphorylated to NDPs and NTPs, which in turn are
  • RNA product per unit energy input e.g., polyphosphate, ATP, or other sources of high energy phosphate.
  • the enzymatic activities are eliminated by removal from the host genome. In some embodiments, the enzymatic activities are eliminated by heat inactivation. In some embodiments, the enzymatic activities are eliminated by protease targeting. In some embodiments the enzymatic activities are eliminated through the use of chemical inhibitors. A combination of any of the foregoing approaches may also be used.
  • Enzymes deleterious to the biosynthesis of RNA may be deleted from the host cell genome during the process of engineering the host cell, provided the enzymes are not essential for host cell ⁇ e.g., bacterial cell) survival and/or growth.
  • Deletion of enzymes or enzyme activities may be achieved, for example, by deleting or modifying in the host cell genome a gene encoding the essential enzyme.
  • An enzyme is "essential for host cell survival” if the enzyme is necessary for the survival of the host cell. That is, if a host cell cannot survive without expression and/or activity of a particular enzyme, then that enzyme is considered essential for host cell survival.
  • an enzyme is "essential for host cell growth” if the enzyme is necessary for the growth of the host cell. That is, if a host cell cannot divide and/or grow without expression and/or activity of a particular enzyme, then that enzyme is considered essential for host cell growth.
  • the enzymes may be heat inactivated.
  • Heat inactivation refers to the process of heating a cell lysate to a temperature sufficient to inactivate (or at least partially inactivate) endogenous nucleases and phosphatases. Generally, the process of heat inactivation involves denaturation of (unfolding of) the deleterious enzyme. The temperature at which endogenous cellular proteins denature varies among organisms. In E. coli, for example, endogenous cellular enzymes generally denature at temperatures above 41 °C.
  • the denaturation temperature may be higher or lower than 41 °C for other organisms.
  • Enzymes of a cell lysate may be heat inactivated at a temperature of 40 °C- 95 °C, or higher.
  • enzymes of a cell lysate may be heat inactivated at a temperature of 40-90 °C, 40-80 °C, 40-70 °C, 40-60 °C, 40-50 °C, 50-80 °C, 50-70 °C, 50-60 °C, 60-80 °C, 60-70 °C, or 70-80 °C.
  • enzymes of a cell lysate may be heat inactivated at a temperature of 40 °C, 42 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C, or 95 °C.
  • enzymes of a cell lysate may be heat inactivated at a temperature of 50-80 °C.
  • enzymes of a cell lysate may be heat inactivated at a temperature of 70 °C.
  • enzymes of a cell lysate may be heat inactivated at a temperature of 60 °C.
  • inhibitors of deleterious enzymes may include, but are not limited to, sodium orthovanadate (inhibitor of protein phosphotyrosyl phosphatases), sodium fluoride (inhibitor of phosphoseryl and phosphothreonyl phosphatases), sodium pyrophosphate (phosphatase inhibitor), sodium phosphate, and/or potassium phosphate.
  • the period of time during which a cell lysate is incubated at elevated temperatures to achieve heat inactivation of endogenous enzymes may vary, depending, for example, on the volume of the cell lysate and the organism from which the cell lysate was prepared.
  • a cell lysate is incubated at a temperature of 35 °C-80 °C for 2 minutes (min) to 48 hours (hr).
  • a cell lysate may be incubated at a temperature of 35 °C-80 °C for 2 min, 4 min, 5 min, 10 min, 15 min, 30 min, 45 min, or 1 hr.
  • a cell lysate is incubated at a temperature of 35 °C-80 °C for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 36, 42, or 48 hr.
  • enzymes are heat inactivated at a temperature of 60-80 °C for 10-20 min. In some embodiments, enzymes are heat inactivated at a temperature of 70 °C for 15 min.
  • enzymes that depolymerize endogenous RNA comprise one or more modifications (e.g., mutations) that render the enzymes more sensitive to heat. These enzymes are referred to as "heat-sensitive enzymes.” Heat-sensitive enzymes denature and become inactivated at temperatures lower than that of their wild-type counterparts, and/or the period of time required to reduce the activity of the heat-sensitive enzymes is shorter than that of their wild-type counterparts.
  • the activity level of a heat-inactivated enzyme may be less than 50% of the activity level of the same enzyme that has not been heat inactivated. In some embodiments, the activity level of a heat-inactivated enzyme is less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, or less than 0.1%) of the activity level of the same enzyme that has not been heat inactivated.
  • an enzyme' s activity may be completely eliminated or reduced.
  • An enzyme is considered completely inactive if the denatured (heat inactivated) form of the enzyme no longer catalyzes a reaction catalyzed by the enzyme in its native form.
  • a heat-inactivated, denatured enzyme is considered "inactivated" when activity of the heat-inactivated enzyme is reduced by at least 50% relative to activity of the enzyme that is not heated (e.g., in its native environment). In some embodiments, activity of a heat-inactivated enzyme is reduced by 50- 100%) relative to the activity of the enzyme that is not heated.
  • activity of a heat- inactivated enzyme is reduced by 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50- 60%), or 50-55%) relative to activity of the enzyme that is not heated.
  • the activity of a heat-inactivated enzyme is reduced by 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% relative to the activity of the enzyme that is not heated.
  • Examples of enzymes that may be heat inactivated, or deleted from the genome of a host cell include, without limitation, nucleases (e.g., RNase III, RNase I, RNase R, P Pase, RNase II, and RNase T), phosphatases (e.g., nucleoside monophosphatase, nucleoside diphosphatase, nucleoside triphosphatase), and other enzymes that depolymerize RNA or dephosphorylate nucleotides.
  • Enzymes that depolymerize RNA include any enzyme that is able to cleave, partially hydrolyze, or completely hydrolyze a RNA molecule.
  • Table 2 provides a list of non-limiting examples of nucleases that may be heat inactivated or, in some instances, deleted from an engineered host cell.
  • Table 3 provides a list of non-limiting examples of phosphatases that may be heat inactivated or, in some instances, deleted from an engineered host cell. Heat inactivation of these and other nucleases and phosphatases is encompassed by the present disclosure.
  • E. coli RNase III preferentially cleaves dsRNA as well as some single-stranded mRNA molecules.
  • the presence of RNase III in cell lysate may limit the accumulation of high concentrations of synthetic RNA ⁇ e.g., dsRNA), because the synthetic RNA is readily cleaved.
  • dsRNA synthetic RNA
  • rnc is essential for cell viability, thus, in some embodiments, rnc is deleted or mutated in engineered host cells.
  • RNase III is heat inactivated following depolymenzation of endogenous RNA.
  • E. coli RNase I localizes to the periplasmic space in intact cells and catalyzes depolymenzation of a wide range of RNA molecules, including rRNA, mRNA, and tRNA. Under physiological conditions the periplasmic localization of this enzyme means that the enzyme has little impact on RNA stability within the cell; however, mixing of the periplasm and cytoplasm in cell lysates permits RNase I access to cellular RNA. The presence of RNasel in a cell lysate may reduce the yield of synthetic RNA through RNA degradation. Neither RNase I nor the gene encoding RNase I, rna, is essential for cell viability, thus, in some embodiments, rna is deleted or mutated in engineered host cells. In other
  • RNase I is heat inactivated following depolymerization of endogenous RNA.
  • E. coli RNase R and RNase T catalyze the depolymerization of dsRNA, rRNA, tRNA, and mRNA, as well as small unstructured RNA molecules. Neither the enzymes nor the genes encoding the enzymes, rnr and rnt, respectively, are essential for cell viability, thus, in some embodiments, rnr and/or rnt are deleted or mutated in engineered host cells (e.g., E. coli host cells). In other embodiments, RNase R and/or RNase T are heat inactivated following the depolymerization of endogenous RNA.
  • E. coli RNase E and PNPase are components of the degradasome, which is responsible for mRNA turnover in cells.
  • RNase E is thought to function together with PNPase and RNase II to turn over cellular mRNA pools. Disruption of the gene encoding RNase E, rne, is lethal in E. coli.
  • RNase E is heat inactivated following depolymerization of endogenous RNA.
  • PNPase nor the gene encoding PNPase, pnp is essential for cell viability, thus, in some embodiments,/? ⁇ is deleted or mutated in engineered host cells (e.g., E. coli host cells).
  • PNPase is heat inactivated following depolymerization of endogenous RNA.
  • E. coli RNase II depolymerizes both mRNA and tRNA in a 3 ' - 5 ' direction. Neither RNase II nor the gene encoding RNase II, rnb, is essential for cell viability, thus, in some embodiments, rnb is deleted or mutated in engineered host cells. In other words, rnb is deleted or mutated in engineered host cells. In other words, rnb is deleted or mutated in engineered host cells. In other
  • RNase II is heat inactivated following depolymerization of endogenous RNA.
  • both PNPase and RNase II are heat inactivated.
  • nucleoside monophosphates NMPs
  • a desired synthetic RNA such as a double-stranded RNA or single-stranded RNA (e.g., mRNA or antisense RNA).
  • a desired synthetic RNA such as a double-stranded RNA or single-stranded RNA (e.g., mRNA or antisense RNA).
  • mRNA or antisense RNA e.g., mRNA or antisense RNA.
  • the phosphates are typically donated from a high-energy phosphate source, such as, for example, phosphoenolpyruvate, ATP, or polyphosphate.
  • the energy source is ATP that is directly added to a cell lysate.
  • the energy source is provided using an ATP regeneration system.
  • polyphosphate and polyphosphate kinase may be used to produce ATP.
  • Other ATP (or other energy) regeneration systems may be used.
  • at least one component of the energy source is added to a cell lysate or cell lysate mixture.
  • a “component” of an energy source includes the substrate(s) and enzyme(s) required to produce energy (e.g., ATP).
  • energy e.g., ATP
  • Non-limiting examples of these components include polyphosphate, polyphosphate kinase, acetyl-phosphate, acetate kinase, phospho-creatine, creatine kinase, phosphoenolpyruvate, and pyruvate kinase.
  • a kinase is an enzyme that catalyzes the transfer of phosphate groups from high- energy, phosphate-donating molecules, such as ATP, to specific substrates/molecules. This process is referred to as phosphorylation, where the substrate gains a phosphate group and the high-energy ATP molecule donates a phosphate group. This transesterification produces a phosphorylated substrate and ADP.
  • the kinases of the present disclosure convert the NMPs to NDPs and NDPs to NTPs.
  • a kinase is a nucleoside monophosphate kinase, which catalyzes the transfer of a high-energy phosphate from ATP to an NMP, resulting in ADP and NDP.
  • nucleoside monophosphate kinases are provided in Tables 4 and 5. As discussed below, thermostable variants of the enzymes listed in Tables 4 and 5 are encompassed by the present disclosure.
  • a cell lysate comprises one or more (or all) of the following four nucleoside monophosphate kinases: thermostable uridylate kinase, thermostable cytidylate kinase, thermostable guanylate kinase and thermostable adenylate kinase.
  • UMP kinase is obtained from Pyrococcus furiosus ⁇ e.g., SEQ ID NO:3 or a variant comprising an amino acid sequence that is at least 70% identical to the amino acid sequence identified by SEQ ID NO:3).
  • CMP kinase is obtained from Thermus thermophilics (e.g., SEQ ID NO:4 or a variant comprising an amino acid sequence that is at least 70% identical to the amino acid sequence identified by SEQ ID NO:4).
  • GMP kinase is obtained from Thermus thermophilics (e.g., SEQ ID NO:4 or a variant comprising an amino acid sequence that is at least 70% identical to the amino acid sequence identified by SEQ ID NO:4).
  • GMP kinase is obtained from Thermus thermophilics (e.g., SEQ ID NO:4 or a variant comprising an amino acid sequence that is at least 70% identical to the amino acid sequence identified by SEQ ID NO:4).
  • GMP kinase is obtained from
  • Thermotoga maritima e.g., SEQ ID NO:5 or a variant comprising an amino acid sequence that is at least 70% identical to the amino acid sequence identified by SEQ ID NO:5.
  • AMP kinase is obtained from Thermus thermophilus ⁇ e.g., SEQ ID NO:6 or a variant comprising an amino acid sequence that is at least 70% identical to the amino acid sequence identified by SEQ ID NO: 6).
  • a NMP kinase has an amino acid sequence identified by the amino acid sequence of any one of SEQ ID NO: 3-6.
  • the NMP kinase has an amino acid sequence that is at least 70% identical to the amino acid sequence of any one of SEQ ID NO: 3-6.
  • the NMP kinase may have an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%), at least 97%, at least 98%, or at least 99% identical to the amino acid sequence identified by any one of SEQ ID NO: 3-6.
  • variants of the enzymes described herein as well as variants of the enzymes ⁇ e.g., "PPK2 variants”).
  • Variant enzymes may share a certain degree of sequence identity with the reference enzyme.
  • identity refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences.
  • Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program ⁇ e.g., "algorithms"). Identity of related molecules can be readily calculated by known methods. "Percent (%) identity” as it applies to amino acid or nucleic acid sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
  • Variants of a particular sequence may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference sequence, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • Techniques for determining identity are codified in publicly available computer programs.
  • Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package (Devereux, J. et al. Nucleic Acids Research, 12(1): 387, 1984), the BLAST suite (Altschul, S. F. et al. Nucleic Acids Res. 25: 3389, 1997), and FASTA (Altschul, S. F. et al. J. Molec. Biol. 215: 403, 1990).
  • Other techniques include: the Smith-Waterman algorithm (Smith, T.F. et al. J. Mol. Biol. 147: 195, 1981; the Needleman-Wunsch algorithm
  • a kinase is a nucleoside diphosphate kinase, which transfers a phosphoryl group to NDP, resulting in NTP.
  • the donor of the phosphoryl group may be, without limitation, ATP, polyphosphate polymer, or phosphoenolpyruvate.
  • Non- limiting examples of kinases that convert NDP to NTP include nucleoside diphosphate kinase, polyphosphate kinase, and pyruvate kinase. As discussed below, thermostable variants of the foregoing enzymes are encompassed by the present disclosure.
  • the NDP kinase(s) is/are obtained from Aquifex aeolicus ⁇ e.g., SEQ ID NO: 9 or a variant comprising an amino acid sequence that is at least 70% identical to the amino acid sequence identified by SEQ ID NO:9).
  • the NDP kinase has an amino acid sequence that is at least 70% identical to the amino acid sequence identified by SEQ ID NO: 9.
  • the NDP kinase may have an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%), or at least 99% identical to the amino acid sequence identified by SEQ ID NO: 9.
  • Phosphorylation of NMPs to NTPs occurs, in some embodiments, through the polyphosphate-dependent kinase pathway ⁇ Figures 2A and 2B), where high-energy phosphate is transferred from polyphosphate to ADP via a polyphosphate kinase (PPK).
  • PPK polyphosphate kinase
  • the polyphosphate kinase belongs to the polyphosphate kinase 1 (PPK1) family, which transfers high-energy phosphate from polyphosphate to ADP to form ATP.
  • This ATP is subsequently used by MP kinases (e.g., AMP kinase, UMP kinase, GMP kinase, and CMP kinase) to convert MPs to their cognate ribonucleotide diphosphates (NDPs). Furthermore, ATP is subsequently used by nucleotide diphosphate kinase to convert NDPs to NTPs. See, e.g., Tables 5 and 6 for exemplary enzymes.
  • MP kinases e.g., AMP kinase, UMP kinase, GMP kinase, and CMP kinase
  • NDPs ribonucleotide diphosphates
  • the polyphosphate kinase belongs to the polyphosphate kinase 2 (PPK2) family.
  • the polyphosphate kinase belongs to a Class I PPK2 family, which transfers high-energy phosphate from polyphosphate to NDPs to form NTPs. ATP produced by the system is used as a high-energy phosphate donor to convert NMPs to NDPs.
  • the polyphosphate kinase belongs to a Class III PPK2 family, which transfers high-energy phosphate from polyphosphate to NMPs and NDPs to form NTPs.
  • Class III PPK2 is used alone to produce NTPs from NMPs.
  • Class III PPK2 is used in combination with other kinases.
  • Class III PPK2 produces ATP from ADP, AMP, and polyphosphate, which is subsequently used by NMP and NDP kinases to convert NMPs to NTPs.
  • PPK2 enzymes for use as provided herein are listed in Table 6 (SEQ ID NO: 8-18).
  • the PPK2 enzymes are listed in Table 6 (SEQ ID NO: 8-18).
  • the PPK2 enzymes are listed in Table 6 (SEQ ID NO: 8-18).
  • the PPK2 enzymes may be thermostable Class III PPK2 enzymes, which favor ATP synthesis over polyphosphate polymerization, and convert both ADP and AMP to ATP.
  • the PPK2 enzymes are used to convert a polyphosphate, such as hexametaphosphate to ATP, at rates ranging, for example, from 10 to 800 mM per hour (e.g., 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 mM per hour).
  • the RNA biosynthesis methods of the present disclosure utilize a PPK2 enzyme that comprises an amino acid sequence identical to the amino acid sequence identified by any one of SEQ ID NO: 8-18.
  • the PPK2 enzyme comprises an amino acid sequence that is at least 70% identical to the amino acid sequence identified by any one of SEQ ID NO: 8-18.
  • the PPK2 enzyme may comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence identified by any one of SEQ ID NO: 8-18.
  • the present disclosure also encompasses fusion enzymes. Fusion enzymes may exhibit multiple activities, each corresponding to the activity of a different enzyme. For example, rather than using an independent nucleoside monophosphate kinase and an independent nucleoside diphosphate kinase, a fusion enzyme (or any other enzyme) having both nucleoside monophosphate kinase activity and nucleoside diphosphate kinase activity may be used.
  • the final step in the biosynthesis of a RNA of interest is the polymerization of NTPs to the RNA (e.g., dsRNA or ssRNA) end product using, for example, a DNA- dependent RNA polymerase.
  • a DNA designed to encode the RNA of interest serves as the template for the synthesis of the RNA of interest.
  • the DNA template may be engineered, in some instances, to have a transcriptional promoter that selectively drives transcription of the RNA of interest.
  • An example DNA template is shown in Figure 3A.
  • the DNA template encodes three RNA domains: a sense domain (domain 1), a flexible hinge domain (domain 2) and a domain complementary to the sense domain (antisense domain 3).
  • the antisense domain binds (hybridizes) to the sense domain to form a double-stranded RNA hairpin stem domain and an adjacent hairpin loop domain.
  • Figures 3B- 3E Other examples of a DNA template are shown in Figures 3B- 3E.
  • the DNA template in Figure 3B contains converging promoter sequences on
  • RNA sequences transcribed from each template strand anneal after transcription.
  • the DNA template in Figure 3C encoded as part of a plasmid, contains converging promoter sequences on complementary strands, as well as one or more terminator sequences to minimize read-through transcription.
  • the DNA template in Figure 3D encoded as part of a plasmid, contains independent promoter-terminator cassettes driving transcription of complementary sequences, which anneal after transcription.
  • the DNA template in Figure 3E encodes a single RNA domain. Use of both DNA-dependent RNA polymerase and RNA- dependent RNA polymerase produces a double-stranded RNA end product.
  • RNA template comprising a
  • RNA polymerase a polymerase for use as provided herein is a single subunit polymerase, is highly selective for its cognate transcriptional promoters, has high fidelity, and is highly efficient.
  • polymerases include, without limitation, T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.
  • Bacteriophage T7 RNA polymerase is a DNA- dependent RNA polymerase that is highly specific for the T7 phage promoters. The 99 KD enzyme catalyzes in vitro RNA synthesis from a cloned DNA sequence under control of the T7 promoter.
  • Bacteriophage T3 RNA polymerase is a DNA-dependent RNA polymerase that is highly specific for the T3 phage promoters. The 99 KD enzyme catalyzes in vitro RNA synthesis from a cloned DNA sequence under the T3 promoter.
  • Bacteriophage SP6 RNA polymerase is a DNA-dependent RNA polymerase that is highly specific for the SP6 phage promoter. The 98.5 KD polymerase catalyzes in vitro RNA synthesis from a cloned DNA template under the SP6 promoter.
  • Each of T7, T3, and SP6 polymerase are optimally active at 37-40°C. In some embodiments, thermostable variants of T7, T3, and SP6 polymerase are used.
  • Thermostable variant polymerases are typically optimally active at temperatures above 40°C (or about 50-60°C).
  • “Conditions that result in production of nucleoside triphosphates and polymerization of the nucleoside triphosphates,” also referred to as “conditions for the biosynthesis of RNA,” may be determined by one of ordinary skill in the art, taking into consideration, for example, optimal conditions for polymerase activity, including pH, temperature, length of time, and salt concentration of the cell lysate as well as any exogenous cofactors.
  • the pH of a cell lysate during the biosynthesis of RNA may have a value of 3.0 to 8.0.
  • the pH value of a cell lysate is 3.0-8.0, 4.0-8.0, 5.0-8.0, 6.0-8.0, 7.0-8.0, 3.0-7.0, 4.0-7.0, 5.0-7.0, 6.0-7.0, 3.0-6.0, 4.0-6.0, 5.0-6.0, 3.0-5.0, 3.0-4.0, or 4.0-5.0.
  • the pH value of a cell lysate is 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.
  • the pH value of a cell lysate during biosynthesis of RNA is 7.0.
  • the temperature of a cell lysate during biosynthesis of RNA may be 15°C to 70 °C.
  • the temperature of a cell lysate during biosynthesis of RNA is 15-60 °C, 15-50 °C, 15-40 °C, 15-30 °C, 25-70 °C, 25-60 °C, 25-50 °C, 25-40 °C, 30-70 °C, 30-60 °C, 30-50 °C, 40-70 °C, 40-60 °C, 40-50 °C, 50-70 °C, or 50-60 °C.
  • the temperature of a cell lysate during biosynthesis of RNA is 15 °C, 25 °C, 32 °C, 37 °C, 42 °C, 45 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, or 70 °C. In some embodiments, the temperature of a cell lysate during biosynthesis of RNA is 50 °C.
  • a cell lysate during biosynthesis of RNA may be incubated for 15 minutes (min) to 72 hours (hrs). In some embodiments, a cell lysate during biosynthesis of RNA is incubated for 30 min-48 hrs. For example, a cell lysate during biosynthesis of RNA may be incubated for 30 min, 45 min, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 18 hrs, 24 hrs, 30 hrs, 36 hrs, 42 hours, or 48 hours. In some embodiments, a cell lysate during biosynthesis of RNA is incubated for 3 hours. In some embodiments, a cell lysate during biosynthesis of RNA is incubated for 24 hours at a temperature of 37 °C.
  • a cell lysate during biosynthesis of RNA is incubated at a pH of 7.0 for 2-4 hours at a temperature of 50 °C.
  • metal ions are added to a cell lysate.
  • metal ions include Mg2 + , Li + , Na + , K + , Ni2 + , Ca2 + , Cu2 + , and Mn2 + .
  • Other metal ions may be used.
  • more than one metal ion may be used.
  • concentration of a metal ion in a cell lysate may be 0.1 mM to 100 mM, or 10 mM to 50 mM.
  • the concentration of a metal ion in a cell lysate is 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5., 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0, 70.0, 80.0, 90.0, or 100.0 mM.
  • salt is added to a cell lysate, for example, to prevent enzyme aggregation.
  • sodium chloride, potassium chloride, sodium acetate, potassium acetate, or a combination thereof may be added to a cell lysate.
  • the concentration of salt in a cell lysate during a RNA depolymerization reaction may be 5 mM to 1 M.
  • the concentration of salt in a cell lysate during a RNA depolymerization reaction 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 500 mM, 750 mM, or 1 M.
  • One advantage of the cell-free RNA-biosynthesis methods of the present disclosure is that all of the enzymes needed to convert endogenous RNA to synthetic double- stranded RNA, for example, may be (but need not be) expressed in a single engineered cell.
  • a clonal population of the engineered cell is cultured to a desired cell density, the cells are lysed, incubated under conditions that result in depolymerization of endogenous RNA to its monomer form ⁇ e.g., at a temperature of 30-37 °C), subjected to temperatures sufficient to inactivate endogenous nucleases and phosphatases ⁇ e.g., 40-90 °C), and incubated under conditions that result in the polymerization of RNA ⁇ e.g., dsRNA or ssRNA) ⁇ e.g., 30-50 °C).
  • the enzymes required for conversion of NMPs to NDPs ⁇ e.g., nucleoside monophosphate kinases and/or polyphosphate kinases), from NDPs to NTPs ⁇ e.g., nucleoside diphosphate kinases and/or polyphosphate kinase), and from NTPs to RNA ⁇ e.g., polymerase
  • NDPs to NTPs e.g., nucleoside diphosphate kinases and/or polyphosphate kinase
  • RNA e.g., polymerase
  • Thermostability refers to the quality of enzymes to resist denaturation at relatively high temperature. For example, if an enzyme is denatured
  • an enzyme having similar activity ⁇ e.g., kinase activity is considered “thermostable” if it does not denature at 42 °C.
  • thermostable enzyme ⁇ e.g., kinase or polymerase
  • an enzyme is considered thermostable if the enzyme (a) retains activity after temporary exposure to high temperatures that denature other native enzymes or (b) functions at a high rate after temporary exposure to a medium to high temperature where native enzymes function at low rates.
  • a thermostable enzyme retains greater than 50% activity following temporary exposure to relatively high temperature (e.g., higher than 41 °C for kinases obtained from E. coli, higher than 37 °C for many RNA polymerases) that would otherwise denature a similar (non-thermostable) native enzyme.
  • thermostable enzyme retains 50-100%) activity following temporary exposure to relatively high temperature that would otherwise denature a similar (non-thermostable) native enzyme.
  • a thermostable enzyme may retain 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%), 50-60%), or 50-55%) activity following temporary exposure to relatively high temperature that would otherwise denature a similar (non-thermostable) native enzyme.
  • thermostable enzyme retains 25%, 30%>, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% activity following temporary exposure to relatively high temperature that would otherwise denature a similar (non-thermostable) native enzyme.
  • the activity of a thermostable enzyme after temporary exposure to medium to high temperature ⁇ e.g., 42-80 °C) is greater than ⁇ e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) greater than) the activity of a similar (non -thermostable) native enzyme.
  • thermostable kinase for example, may be measured by the amount of MP or NDP the kinase is able to phosphorylate.
  • a thermostable kinase, at relatively high temperature e.g., 42 °C converts greater than 50% of NMP to NDP, or greater than 50% of NDP to NTP, in the same amount of time required to complete a similar conversion at 37 °C.
  • thermostable kinase, at relatively high temperature converts greater than 60% of NMP to NDP, or greater than 60% of NDP to NTP, in the same amount of time required to complete a similar conversion at 37 °C.
  • a thermostable kinase, at relatively high temperature converts greater than 70% of NMP to NDP, or greater than 70% of NDP to NTP, in the same amount of time required to complete a similar conversion at 37 °C.
  • thermostable kinase, at relatively high temperature converts greater than 80% of NMP to NDP, or greater than 80% of NDP to NTP, in the same amount of time required to complete a similar conversion at 37 °C.
  • a thermostable kinase, at relatively high temperature converts greater than 90% of NMP to NDP, or greater than 90% of NDP to NTP, in the same amount of time required to complete a similar conversion at 37 °C.
  • thermostable polymerase for example, is assessed based on fidelity and polymerization kinetics (e.g., rate of polymerization).
  • one unit of a thermostable T7 polymerase for example, may incorporate 10 nmoles of NTP into acid insoluble material in 30 minutes at temperatures above 37 °C (e.g., at 50 °C).
  • Thermostable enzymes may remain active (able to catalyze a reaction) at a temperature of 42 °C to 80 °C, or higher.
  • thermostable enzymes remain active at a temperature of 42-80 °C, 42-70 °C, 42-60 °C, 42-50 °C, 50-80 °C, 50-70 °C, 50-60 °C, 60-80 °C, 60-70 °C, or 70-80 °C.
  • thermostable enzymes may remain active at a temperature of 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, 70 °C, 71 °C, 72 °C, 73 °C, 74 °C, 75 °C, 76 °C, 77 °C, 78 °C, 79 °C, or 80 °C.
  • thermostable enzymes may remain active at relatively high temperatures for 15 minutes to 48 hours, or longer.
  • thermostable enzymes may remain active at relatively high temperatures for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 36, 42, or 48 hours.
  • thermostable NMP kinases are listed in Tables 5 and 7.
  • Other thermostable kinases include thermostable nucleoside diphosphate kinases, thermostable pyruvate kinases, and thermostable polyphosphate kinases (see, e.g., Table 6).
  • Other thermostable kinases are encompassed by the present disclosure.
  • RNA polymerases are listed in Table 8. Other RNA polymerases, including thermostable RNA polymerases, are encompassed by the present disclosure.
  • thermostable T7 RNA polymerases may include one or more of the following point mutations: V426L, A702V, V795I, S430P, F849I, S633I, F880Y, C510R, and S767G
  • a variant thermostable T7 RNA polymerase includes V426L, A702V, and V795I mutations. In some embodiments, a variant thermostable T7 RNA polymerase includes S430P, F849I, S633I, and F880Y mutations. In some embodiments, a variant thermostable T7 RNA polymerase includes F880Y, S430P, F849I, S633I, C510R, and S767G mutations. In some embodiments, a variant thermostable T7 RNA polymerase includes Y639V, H784G, E593G, and V685A mutations. In some embodiments, a variant
  • thermostable T7 RNA polymerase includes S430P, N433T, S633P, F849I, and F880Y mutations. Other variant and recombinant thermostable polymerases are encompassed by the present disclosure.
  • thermostable T7 polymerase is used to produce a RNA of interest.
  • a thermostable T7 polymerase e.g., incubated at a temperature of 40- 60 °C
  • concentration of 1-2% total protein may be used to synthesize RNA of interest at a rate of greater than 2 g/L/hr (or, e.g., 2 g/L/hr - 10 g/L/hr).
  • thermostable T7 polymerase e.g., incubated at a temperature of 40-60 °C having a concentration of 3-5% total protein may be used to synthesize RNA of interest at a rate of greater than 10 g/L/hr (or, e.g., 10 g/L/hr - 20 g/L/hr).
  • thermostable polymerases/enzymes other enzymes/polymerases may be used.
  • polymerase may be exogenously added to heat-inactivated cell lysates, for example, to compensate for any reduction or loss of activity of the thermostable enzyme(s).
  • RNA of interest may be single- stranded or double-stranded.
  • the RNA is a double-stranded RNA interference molecule.
  • a RNA of interest may be an siRNA or a hairpin RNA interference molecule.
  • a RNA of interest is encoded by a DNA template, examples of which are shown in Figures 3A-3E.
  • the RNA produced using the template of Figure 3A includes a sense domain (domain 1), a flexible hinge domain (domain 2) and a domain complementary to the sense domain (antisense domain 3). Following transcription of the DNA template, the antisense domain binds (hybridizes) to the sense domain to form a double-stranded RNA hairpin stem domain and an adjacent hairpin loop (hinge) domain.
  • a double-stranded hairpin stem domain is formed by the binding of two complementary nucleic acid domains (e.g., discrete nucleotide sequences) to each other. Nucleic acid domains are "complementary” if they bind (base pair via Watson-Crick interactions, hybridize) to each other to form a double-stranded nucleic acid.
  • complementary domains of a DNA template encoding a RNA of interest may vary, depending, for example, on the desired end product.
  • Complementary domains may have a length of, for example, 4 to 1000 nucleotides, or longer.
  • complementary domains may have a length of 4 to 10, 4 to 20, 4 to 30, 4 to 50, 4 to 60, 4 to 70, 4 to 80, 4 to 90, 4 to 100, 4 to 200, 4 to 300, 4 to 400, or 4 to 500, or 4 to 1000 nucleotides.
  • complementary domains have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
  • complementary domains have a length of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides.
  • a hairpin loop domain is also formed by binding of two complementary nucleic acid domains.
  • a hairpin loop domain is the intervening sequence between two
  • a hairpin loop domain is non-specific, meaning that it is not designed to bind intramolecularly or to another nucleic acid.
  • a hairpin loop domain forms a loop-like structure upon binding of the complementary domains to form a double- stranded hairpin stem domain.
  • a hairpin loop domain has a length of 4 to 500 nucleotides, or more.
  • a hairpin loop domain may have a length of 4 to 10, 4 to 20, 4 to 30, 4 to 50, 4 to 60, 4 to 70, 4 to 80, 4 to 90, 4 to 100, 4 to 200, 4 to 300, 4 to 400, or 4 to 500 nucleotides.
  • a hairpin loop domain has a length of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides.
  • a "double-stranded RNA" of the present disclosure encompasses wholly double- stranded molecules, which do not contain a single-stranded region (e.g., a loop or overhang), as well as partially double-stranded molecules, which contain a double-stranded region and a single-stranded region (e.g., a loop or overhang).
  • the dsRNA product depicted at the bottom of Figure 3A is considered a partially double-stranded molecule
  • the dsRNA product depicted at the bottom of Figure 3B is considered a wholly double-stranded molecule.
  • RNA messenger RNA
  • antisense RNA examples include methods of synthesizing mRNA and other single-stranded RNA molecules.
  • These methods may comprise (a) lysing cultured engineered cells that comprise RNA, an enzyme that depolymerizes RNA, thermostable kinases, a thermostable RNA polymerase, thereby producing a cell lysate, (b) incubating the cell lysate produced in step (a) under conditions that result in depolymerization of RNA, thereby producing a cell lysate that comprises nucleoside monophosphates, (c) heating the cell lysate produced in step (b) to a temperature that inactivates endogenous nucleases and phosphatases without inactivating the thermostable kinases and thermostable RNA polymerase, thereby producing a cell lysate that comprises heat-inactivated nucleases and phosphatases, and (d) incubating the cell lysate produced in (c) in the presence of an energy source and an engineered DNA template containing a promoter operably linked to a nucleotide sequence
  • such methods may comprise (a) combining cell ly sates obtained from engineered cells that comprise endogenous, polymeric RNA, an enzyme that depolymerizes RNA, thermostable nucleoside monophosphate (NMP) kinases, thermostable nucleoside diphosphate (NDP) kinases, a thermostable PPK2 kinase, and/or a polyphosphate, to produce a cell lysate mixture, (b) incubating the cell lysate mixture produced in step (a) under conditions that result in depolymerization of RNA, thereby producing a cell lysate that comprises nucleoside monophosphates, (c) heating the cell lysate produced in step (b) to a temperature that inactivates phosphatases and RNases (and any other activities that may be detrimental to RNA stability or polymerization fidelity, such as native RNA polymerase, NMP reductases, and/or nucleosidases
  • the DNA template encoding the RNA containing a single target domain is transcribed using a DNA-dependent RNA polymerase, such as, for example, a T7 RNA polymerase, and the resulting RNA transcript serves as a template for a RNA-dependent RNA polymerase, such as, for example, the phage ⁇ 6 RdRP, to synthesize a complementary RNA molecule, yielding a dsRNA.
  • a DNA-dependent RNA polymerase such as, for example, a T7 RNA polymerase
  • a RNA-dependent RNA polymerase such as, for example, the phage ⁇ 6 RdRP
  • This phage encodes an RdRP that is capable of synthesizing RNA using a RNA template, yielding a dsRNA molecule.
  • the ⁇ 6 RdRP is capable of polymerizing RNA absent a primer molecule, thus the polymerase requires only template RNA (Wright, S. et al, 2012. Journal of Virology. Mar;86(5):2837-49; Van Dijk, AA., et al, 2004. J Gen Virol. May;85(Pt 5), incorporated herein by reference).
  • Other RNA-dependent RNA polymerase (RdRP) are encompassed by the present disclosure.
  • the engineered cells comprise a DNA template encoding the RNA of interest.
  • a DNA template encoding the RNA may be integrated into the genomic DNA of the engineered cells, or a DNA template may be introduced into the engineered cells on a plasmid.
  • the DNA template is added to the cell lysate during biosynthesis of the RNA of interest ⁇ e.g., following a heat inactivation step).
  • the concentration of the DNA template in a cell lysate is 0.05-1 ⁇ g/ ⁇ l.
  • the concentration of the DNA template in a cell lysate is 0.05 ⁇ g/ ⁇ l, 0.1 ⁇ g/ ⁇ l, 0.5 ⁇ / ⁇ 1, 1.0 ⁇ ⁇ / ⁇ 1.
  • RNA end products of interest include messenger RNA (mRNA) and short/small-interfering RNA (siRNA) (a synthetic RNA duplex designed to specifically target a particular mRNA for degradation).
  • mRNA messenger RNA
  • siRNA short/small-interfering RNA
  • the concentration of RNA end product (biosynthesized
  • RNA of interest is at least 1 g/L to 50 g/L of cell lysate.
  • concentration of RNA of interest is at least 1 g/L to 50 g/L of cell lysate.
  • RNA end product may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g/L, or more.
  • RNA of interest is designed to bind to a target nucleic acid of interest and is used, for example, as a therapeutic, prophylactic, or diagnostic agent.
  • Engineered cells of the present disclosure may express ⁇ e.g., endogenously express) enzymes necessary for the health of the cells that may have a negative impact on the production of nucleic acids, such as RNA.
  • Such enzymes are referred to herein as "target enzymes.”
  • target enzymes expressed by engineered cells may compete for substrates or cofactors with an enzyme that increases the rate of precursor supplied to a RNA biosynthetic pathway.
  • target enzymes expressed by the engineered cells may compete for substrates or cofactors with an enzyme that is a key pathway entry enzyme of a RNA biosynthetic pathway.
  • target enzymes expressed by the engineered cells may compete for substrates or cofactors with an enzyme that supplies a substrate or cofactor of a RNA biosynthetic pathway.
  • target enzymes can be modified to include a site-specific protease-recognition sequence in their protein sequence such that the target enzyme may be "targeted” and cleaved for inactivation during RNA production ⁇ see, e.g., U.S. Publication No. 2012/0052547 Al, published on March 1, 2012; and International Publication No. WO 2015/021058 A2, published February 12, 2015, each of which is incorporated by reference herein).
  • Cleavage of a target enzyme containing a site-specific protease-recognition sequence results from contact with a cognate site-specific protease is sequestered in the periplasm of cell (separate from the target enzyme) during the cell growth phase ⁇ e.g., as engineered cells are cultured) and is brought into contact with the target enzyme during the RNA production phase ⁇ e.g., following cell lysis to produce a cell lysate).
  • engineered cells of the present disclosure comprise, in some embodiments, (i) an engineered nucleic acid encoding a target enzyme that negatively impacts the rate of RNA production and includes a site-specific protease-recognition sequence in the protein sequence of the target enzyme, and (ii) an engineered nucleic acid encoding a site-specific protease that cleaves the site-specific protease-recognition sequence of the target enzyme and includes a periplasmic-targeting sequence.
  • This periplasmic-targeting sequence is responsible for sequestering the site- specific protease to the periplasmic space of the cell until the cell is lysed. Examples of periplasmic-targeting sequences are provided below.
  • proteases examples include, without limitation, alanine carboxypeptidase, proteases obtained from Armillaria mellea, astacin, bacterial leucyl aminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosol alanyl aminopeptidase, elastase, endoproteinase Brg-C, enterokinase, gastricsin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, Iga-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionyl aminopeptidase, myxo
  • Enzymes of a nucleic acid ⁇ e.g., RNA) biosynthetic pathway may include at least one enzyme that has a negative impact on the health ⁇ e.g., viability) of a cell.
  • an enzyme can be modified to include a relocation sequence such that the enzyme is relocated to a cellular or extra-cellular compartment where it is not naturally located and where the enzyme does not negatively impact the health of the cell ⁇ see, e.g., Publication No. US-2011-0275116-A1, published on November 10, 2011, incorporated by reference herein).
  • an enzyme of a biosynthetic pathway may be relocated to the periplasmic space of a cell.
  • engineered cells of the present disclosure comprise at least one enzyme of a nucleic acid ⁇ e.g., RNA) biosynthetic pathway that is linked to a periplasmic-targeting sequence.
  • a periplasmic-targeting sequence is an amino acid sequence that targets to the periplasm of a cell the protein to which it is linked.
  • a protein that is linked to a periplasmic-targeting sequence will be sequestered in the periplasm of the cell in which the protein is expressed.
  • Periplasmic-targeting sequences may be derived from the N-terminus of bacterial secretory protein, for example. The sequences vary in length from about 15 to about 70 amino acids.
  • the primary amino acid sequences of periplasmic-targeting sequences vary, but generally have a common structure, including the following components: (i) the N-terminal part has a variable length and generally carries a net positive charge; (ii) following is a central hydrophobic core of about 6 to about 15 amino acids; and (iii) the final component includes four to six amino acids which define the cleavage site for signal peptidases.
  • Periplasmic-targeting sequences of the present disclosure may be derived from a protein that is secreted in a Gram negative bacterium.
  • the secreted protein may be encoded by the bacterium, or by a bacteriophage that infects the bacterium.
  • Gram negative bacterial sources of secreted proteins include, without limitation, members of the genera Escherichia, Pseudomonas, Klebsiella, Salmonella, Caulobacter, Methylomonas, Acetobacter, Achromobacter, Acinetobacter, Aeromonas, Agrobacterium, Alcaligenes, Azotobacter, Burkholderia, Citrobacter, Comamonas, Enterobacter, Erwinia, Rhizobium, Vibrio, and Xanthomonas.
  • periplasmic-targeting sequences for use in accordance with the present disclosure include, without limitation, sequences selected from the group consisting of: MKIKTGARILALSALTTMMFSASALA (SEQ ID NO: 19);
  • Engineered cells of the present disclosure typically comprise at least one, most, or all, of the enzymatic activities required to biosynthesize RNA.
  • Engineered cells are cells that comprise at least one engineered ⁇ e.g., recombinant or synthetic) nucleic acid, or are otherwise modified such that they are structurally and/or functionally distinct from their naturally-occurring counterparts. Thus, a cell that contains an engineered nucleic acid is considered an "engineered cell.”
  • Engineered cells of the present disclosure comprise RNA, enzymes that depolymerizes RNA, thermostable kinases, and/or thermostable polymerases.
  • the engineered cells further comprise a DNA template containing a promoter operably linked to a nucleotide sequence encoding a RNA of interest.
  • Engineered cells express selectable markers.
  • Selectable markers are typically used to select engineered cells that have taken up and expressed an engineered nucleic acid following transfection of the cell (or following other procedure used to introduce foreign nucleic acid into the cell).
  • a nucleic acid encoding product may also encode a selectable marker.
  • selectable markers include, without limitation, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics (e.g., ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, tetracycline resistance genes and chloramphenicol resistance genes) or other compounds.
  • Additional examples of selectable markers include, without limitation, genes encoding proteins that enable the cell to grow in media deficient in an otherwise essential nutrient (auxotrophic markers). Other selectable markers may be used in accordance with the present disclosure.
  • An engineered cell "expresses" a product if the product, encoded by a nucleic acid (e.g., an engineered nucleic acid), is produced in the cell.
  • a nucleic acid e.g., an engineered nucleic acid
  • gene expression refers to the process by which genetic instructions in the form of a nucleic acid are used to synthesize a product, such as a protein (e.g., an enzyme).
  • Engineered cells may be prokaryotic cells or eukaryotic cells. In some
  • engineered cells are bacterial cells, yeast cells, insect cells, mammalian cells, or other types of cells.
  • Engineered bacterial cells of the present disclosure include, without limitation, engineered Escherichia spp., Streptomyces spp., Zymomonas spp., Acetobacter spp.,
  • Citrobacter spp. Synechocystis spp., Rhizobium spp., Clostridium spp., Coryne bacterium spp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia spp.,
  • Acidithiobacillus spp. Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp., Thermus spp.,
  • Stenotrophomonas spp. Chromobacterium spp., Sinorhizobium spp., Saccharopolyspora spp., Agrobacterium spp., and Pantoea spp.
  • Engineered yeast cells of the present disclosure include, without limitation, engineered Saccharomyces spp., Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia and Pichia.
  • engineered cells of the present disclosure are engineered Escherichia coli cells, Bacillus subtilis cells, Pseudomonas putida cells, Saccharomyces cerevisae cells, or Lactobacillus brevis cells. In some embodiments, engineered cells of the present disclosure are engineered Escherichia coli cells. Engineered Nucleic Acids
  • nucleic acid is at least two nucleotides covalently linked together, and in some instances, may contain phosphodiester bonds ⁇ e.g., a phosphodiester "backbone"). Nucleic acids ⁇ e.g., components, or portions, of nucleic acids) may be naturally occurring or engineered. “Naturally occurring” nucleic acids are present in a cell that exists in nature in the absence of human intervention. “Engineered nucleic acids” include recombinant nucleic acids and synthetic nucleic acids.
  • a “recombinant nucleic acid” refers to a molecule that is constructed by joining nucleic acid molecules ⁇ e.g., from the same species or from different species) and, typically, can replicate in a living cell.
  • a “synthetic nucleic acid” refers to a molecule that is biologically synthesized, chemically synthesized, or by other means synthesized or amplified.
  • a synthetic nucleic acid includes nucleic acids that are chemically modified or otherwise modified but can base pair with naturally-occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.
  • Engineered nucleic acids may contain portions of nucleic acids that are naturally occurring, but as a whole, engineered nucleic acids do not occur naturally and require human intervention.
  • a nucleic acid encoding a product of the present disclosure is a recombinant nucleic acid or a synthetic nucleic acid. In other embodiments, a nucleic acid encoding a product is naturally occurring.
  • An engineered nucleic acid encoding RNA may be operably linked to a "promoter,” which is a control region of a nucleic acid at which initiation and rate of transcription of the remainder of a nucleic acid are controlled.
  • a promoter drives expression or drives transcription of the nucleic acid that it regulates.
  • a promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as "endogenous.”
  • a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded sequence in its natural environment.
  • promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring" such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR).
  • a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to the nucleic acid it regulates to control ("drive") transcriptional initiation and/or expression of that nucleic acid.
  • Engineered nucleic acids of the present disclosure may contain a constitutive promoter or an inducible promoter.
  • a "constitutive promoter” refers to a promoter that is constantly active in a cell.
  • An “inducible promoter” refers to a promoter that initiates or enhances transcriptional activity when in the presence of, influenced by, or contacted by an inducer or inducing agent, or activated in the absence of a factor that causes repression.
  • Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art.
  • inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol -regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature/heat-inducible, phosphate-regulated (e.g., PhoA), and light-regulated promoters.
  • An inducer or inducing agent may be endogenous or a normally exogenous condition (e.g., light), compound (e.g., chemical or non-chemical compound) or protein that contacts an inducible promoter in such a way as to be active in regulating transcriptional activity from the inducible promoter.
  • a signal that regulates transcription refers to an inducer signal that acts on an inducible promoter.
  • a signal that regulates transcription may activate or inactivate transcription, depending on the regulatory system used. Activation of transcription may involve directly acting on a promoter to drive transcription or indirectly acting on a promoter by inactivation a repressor that is preventing the promoter from driving transcription. Conversely, deactivation of transcription may involve directly acting on a promoter to prevent transcription or indirectly acting on a promoter by activating a repressor that then acts on the promoter.
  • Engineered nucleic acids may be introduced into host cells using any means known in the art, including, without limitation, transformation, transfection (e.g., chemical (e.g., calcium phosphate, cationic polymers, or liposomes) or non-chemical (e.g.,
  • transfection e.g., transfection
  • transduction e.g., viral transduction
  • Enzymes or other proteins encoded by a naturally-occurring, intracellular nucleic acid may be referred to as "endogenous enzymes" or “endogenous proteins.”
  • engineered cells are cultured. “Culturing” refers to the process by which cells are grown under controlled conditions, typically outside of their natural environment.
  • engineered cells such as engineered bacterial cells, may be grown as a cell suspension in liquid nutrient broth, also referred to as liquid "culture medium.”
  • Examples of commonly used bacterial Escherichia coli growth media include, without limitation, LB (Lysogeny Broth) Miller broth (1% NaCl): 1% peptone, 0.5% yeast extract, and 1% NaCl; LB (Lysogeny Broth) Lennox Broth (0.5% NaCl): 1% peptone, 0.5% yeast extract, and 0.5% NaCl; SOB medium (Super Optimal Broth): 2% peptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KC1, 10 mM MgCl 2 , 10 mM MgS0 4 ; SOC medium (Super Optimal broth with Catabolic repressor): SOB + 20 mM glucose; 2x YT broth (2x Yeast extract and Tryptone): 1.6% peptone, 1% yeast extract, and 0.5% NaCl; TB (Terrific Broth) medium: 1.2% peptone, 2.4% yeast extract, 72
  • Examples of high density bacterial Escherichia coli growth media include, but are not limited to, DNAGroTM medium, ProGroTM medium, AutoXTM medium, DetoXTM medium, InduXTM medium, and SecProTM medium.
  • engineered cells are cultured under conditions that result in expression of enzymes or nucleic acids. Such culture conditions may depend on the particular product being expressed and the desired amount of the product.
  • engineered cells are cultured at a temperature of 30 °C to 40 °C.
  • engineered cells may be cultured at a temperature of 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C or 40 °C.
  • engineered cells such as engineered E. coli cells, are cultured at a temperature of 37 °C.
  • engineered cells are cultured for a period of time of 12 hours to 72 hours, or more.
  • engineered cells may be cultured for a period of time of 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours.
  • engineered cells such as engineered bacterial cells, are cultured for a period of time of 12 to 24 hours.
  • engineered cells are cultured for 12 to 24 hours at a temperature of 37 °C.
  • engineered cells are cultured ⁇ e.g., in liquid cell culture medium) to an optical density, measured at a wavelength of 600 nm (OD600), of 5 to 200. In some embodiments, engineered cells are cultured to an OD600 of 5, 10, 15, 20, 25, 50, 75, 100, 150, or 200.
  • engineered cells are cultured to a density of 1 x 10 8 (OD ⁇ 1) to 2 x 10 11 (OD ⁇ 200) viable cells/ml cell culture medium.
  • OD ⁇ 1 x 10 8 OD ⁇ 1
  • 2 x 10 11 OD ⁇ 200
  • engineered cells are cultured in a bioreactor.
  • a bioreactor refers simply to a container in which cells are cultured, such as a culture flask, a dish, or a bag that may be single-use (disposable), autoclavable, or sterilizable.
  • the bioreactor may be made of glass, or it may be polymer-based, or it may be made of other materials.
  • bioreactors include, without limitation, stirred tank (e.g., well mixed) bioreactors and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibromixers, fluidized bed reactors, and membrane bioreactors.
  • the mode of operating the bioreactor may be a batch or continuous processes and will depend on the engineered cells being cultured.
  • a bioreactor is continuous when the feed and product streams are continuously being fed and withdrawn from the system.
  • a batch bioreactor may have a continuous recirculating flow, but no continuous feeding of nutrient or product harvest.
  • cells are inoculated at a lower viable cell density in a medium that is similar in composition to a batch medium. Cells are allowed to grow exponentially with essentially no external manipulation until nutrients are somewhat depleted and cells are approaching stationary growth phase. At this point, for an intermittent harvest batch-fed process, a portion of the cells and product may be harvested, and the removed culture medium is replenished with fresh medium. This process may be repeated several times. For production of recombinant proteins and antibodies, a fed-batch process may be used.
  • RNA e.g., ssRNA or dsRNA
  • engineered cells may be grown in liquid culture medium in a volume of 5 liters (L) to 250,000 L, or more. In some embodiments, engineered cells may be grown in liquid culture medium in a volume of greater than (or equal to) 10 L, 100 L, 1000 L, 10000 L, or 100000 L. In some embodiments, engineered cells are grown in liquid culture medium in a volume of 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, 100 L, 500 L, 1000 L, 5000 L, 10000 L, 100000 L, 150000 L, 200000 L, 250000 L, or more.
  • engineered cells may be grown in liquid culture medium in a volume of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to 20 L, 10 L to 25 L, 20 L to 30 L, 10 L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25 L, 15 L to 30 L, 15 L to 35 L, 15 L to 40 L, 15 L to 45 L, or 15 to 50 L.
  • engineered cells may be grown in liquid culture medium in a volume of 100 L to 300000 L, 100 L to 200000 L, or 100 L to 100000 L.
  • culturing of engineered cells is followed by lysing the cells.
  • “Lysing” refers to the process by which cells are broken down, for example, by viral, enzymatic, mechanical, or osmotic mechanisms.
  • a “cell lysate” refers to a fluid containing the contents of lysed cells (e.g., lysed engineered cells), including, for example, organelles, membrane lipids, proteins, nucleic acids and inverted membrane vesicles. Cell lysates of the present disclosure may be produced by lysing any population of engineered cells, as provided herein.
  • lysing Methods of cell lysis, referred to as “lysing,” are known in the art, any of which may be used in accordance with the present disclosure. Such cell lysis methods include, without limitation, physical lysis such as homogenization.
  • protease inhibitors and/or phosphatase inhibitors may be added to the cell lysate or cells before lysis, or these activities may be removed by heat inactivation, gene inactivation, or protease targeting.
  • Cell lysates in some embodiments, may be combined with at least one nutrient.
  • cell lysates may be combined with Na 2 HP0 4 , KH 2 P0 4 , H 4 C1, NaCl, MgS0 4 , CaCl 2 .
  • Examples of other nutrients include, without limitation, magnesium sulfate, magnesium chloride, magnesium orotate, magnesium citrate, potassium phosphate monobasic, potassium phosphate dibasic, potassium phosphate tribasic, sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate tribasic, ammonium phosphate monobasic, ammonium phosphate dibasic, ammonium sulfate, ammonium chloride, and ammonium hydroxide.
  • Cell lysates in some embodiments, may be combined with at least one cofactor.
  • cell lysates may be combined with adenosine diphosphate (ADP), adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD+), or other non-protein chemical compounds required for activity of an enzyme (e.g., inorganic ions and coenzymes).
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • NAD+ nicotinamide adenine dinucleotide
  • other non-protein chemical compounds required for activity of an enzyme e.g., inorganic ions and coenzymes.
  • cell lysates are incubated under conditions that result in RNA depolymerization. In some embodiments, cell lysates are incubated under conditions that result in production of ssRNA or dsRNA.
  • the volume of cell lysate used for a single reaction may vary. In some
  • the volume of a cell lysate is 0.001 to 250 m 3 .
  • the volume of a cell lysate may be 0.001 m 3 , 0.01 m 3 , 0.1 m 3 , 1 m 3 , 5 m 3 , 10 m 3 , 15 m 3 , 20 m 3 , 25 m 3 , 30 m 3 , 35 m 3 , 40 m 3 , 45 m 3 , 50 m 3 , 55 m 3 , 60 m 3 , 65 m 3 , 70 m 3 , 75 m 3 , 80 m 3 , 85 m 3 , 90 m 3 , 95 m 3 , 100 m 3 , 105 m 3 , 110 m 3 , 115 m 3 , 120 m 3 , 125 m 3 , 130 m 3 , 135 m 3 , 140 m 3 , 145 m 3 , 150 m 3 , 155
  • the volume of a cell lysate is 25 m to 250 m , 50 m to 250 m , or 100 m to 250 m 3 .
  • dsRNA e.g., dsRNA, ssRNA
  • a concentration of 1-50 g/L e.g., 30, 35, 40, 45, or 50 g/L.
  • Downstream processing increases purity to as much as 99% (e.g., 75, 80, 85, 90, 95, 96, 97, 98, or 99%) dsRNA by weight.
  • An example of downstream processing is shown in Figure 4, starting with the addition of a protein precipitating agent (e.g., ammonium acetate) followed by disc-stack centrifugation (DSC) to remove protein, lipids, and some DNA from the product stream. Ultrafiltration is then implemented to remove salts and volume.
  • a protein precipitating agent e.g., ammonium acetate
  • DSC disc-stack centrifugation
  • RNA product which is subsequently separated from the bulk liquid using disc stack centrifugation, for example, yielding an -80% purity RNA product stream. Further chromatographic polishing yield a -99% pure product.
  • RNA biosynthesizing ribonucleic acid
  • step (b) incubating the cell lysate produced in step (a) under conditions that result in depolymerization of RNA, thereby producing a cell lysate that comprises nucleoside monophosphates;
  • step (c) heating the cell lysate produced in step (b) to a temperature that inactivates endogenous nucleases and phosphatases without inactivating the thermostable kinases and thermostable RNA polymerase, thereby producing a cell lysate that comprises heat- inactivated nucleases and phosphatases;
  • RNA of the engineered cells of step (a) is endogenous RNA.
  • RNA comprises ribosomal RNA, messenger RNA, transfer RNA, or a combination thereof.
  • cultured engineered cells comprise at least two enzymes that depolymerize RNA.
  • RNA is selected from the group consisting of: SI nuclease, Nuclease PI, RNase II, RNase III, RNase R, RNase JI, NucA, PNPase, RNase T, RNase E, RNaseG and combinations thereof.
  • step (b) comprises a Mg 2+" chelating agent.
  • EDTA ethylenediaminetetraacetic acid
  • thermostable kinases comprise thermostable nucleoside monophosphate kinases.
  • thermostable nucleoside monophosphate kinases are selected from the group consisting of thermostable uridylate kinases, thermostable cytidylate kinases, thermostable guanylate kinases, and thermostable adenylate kinases.
  • the stable nucleoside monophosphate kinases a selected from the group consisting of a thermostable Pyrococcus furiosus uridylate kinase encoded by a pyrH gene (PfPyrH), a thermostable Thermus thermophilics adenylate kinase encoded by a adk gene (TthAdk), a thermostable Thermus thermophilus cytidylate kinase encoded by a cmk gene (TthCmk), and a thermostable Pyrococcus furiosus guanylate kinase encoded by a gmk gene (PfGmk).
  • PfPyrH thermostable Pyrococcus furiosus uridylate kinase encoded by a pyrH gene
  • TthAdk thermostable Thermus thermophilics adenylate kinase encoded by a adk gene
  • thermostable kinases comprise thermostable nucleoside diphosphate kinases.
  • thermostable nucleoside diphosphate kinases are selected from the group consisting of thermostable nucleoside phosphate kinases, thermostable pyruvate kinases, and thermostable polyphosphate kinases.
  • thermostable nucleoside diphosphate kinases is a thermostable Aquifex aeolicus enzyme encoded by a ndk gene. 22. The method of any one of paragraphs 1-21, wherein the cells comprise a thermostable nucleoside monophosphate kinase and a thermostable nucleoside diphosphate kinase.
  • thermostable uridylate kinase thermostable cytidylate kinase, thermostable guanylate kinase, thermostable adenylate kinase, and thermostable polyphosphate kinase.
  • thermostable RNA polymerase is a thermostable DNA-dependent RNA polymerase.
  • thermostable T7 RNA polymerases thermostable SP6 RNA polymerases
  • thermostable T3 RNA polymerases thermostable T3 RNA polymerases
  • step (c) is at least 50°C.
  • step (c) is at 50°C-80°C.
  • step (c) comprises heating the cell lysate for at least 15 minutes.
  • step (c) comprises heating the cell lysate to a temperature of at least 65°C for 15 minutes.
  • step (d) The method of any one of paragraphs 1-30, wherein the nucleoside triphosphates in step (d) are produced at a rate of 15-30 mM/hour.
  • RNA of interest produced in step (d) is double-stranded RNA.
  • RNA of interest produced in step (d) is an mRNA containing complementary domains linked by a hinged domain.
  • RNA of interest produced in step (d) is produced at a concentration of at least 4 g/L, at least 6 g/L, at least 6 g/L, or at least 10 g/L.
  • the method of paragraph 35 further comprising purifying the double- stranded RNA.
  • the purifying step comprises combining the cell lysate of step (d) with a protein precipitating agent and removing precipitated protein, lipids, and DNA.
  • An engineered cell comprising RNA, an enzyme that depolymerizes RNA, a thermostable kinase and a thermostable RNA polymerase.
  • the engineered cell of paragraph 39 further comprising an engineered DNA template containing a promoter operably linked to a nucleotide sequence encoding a RNA of interest.
  • a method comprising:
  • the method of paragraph 43 further comprising incubating the cell lysate under conditions that result in depolymerization of RNA to produce a cell lysate that comprises nucleoside monophosphates.
  • the method of paragraph 44 further comprising heating the cell lysate to a temperature that inactivates endogenous nucleases and phosphatases without inactivating the thermostable kinases and thermostable RNA polymerase to produce a cell lysate that comprises heat-inactivated nucleases and phosphatases.
  • the method of paragraph 45 further comprising incubating the cell lysate that comprises heat-inactivated nucleases and phosphatases in the presence of an energy source and an engineered DNA template containing a promoter operably linked to a nucleotide sequence encoding a RNA of interest, under conditions that result in production of nucleoside triphosphates and polymerization of the nucleoside triphosphates, to produce a cell lysate that comprises the RNA of interest.
  • nuclease(s) for digesting lysate RNA
  • a series of screening experiments were performed using commercially-available enzymes chosen based on their ability to generate 5'-NMPs or oligonucleotides. The activity of these enzymes was first determined using purified E. coli RNA and reaction conditions recommended by the manufacturer, where RNA depolymerization was monitored by the release of acid-soluble nucleotides. Under these conditions, four nucleases demonstrated depolymerization activity over background. The endonucleases Benzonase and RNase A, which served as positive controls, yielded immediate conversion of RNA to acid-soluble nucleotides ⁇ Figure 5A).
  • RNA with the exonucleases PI and RNase R yielded a time-dependent conversion of RNA to acid-soluble nucleotides, with RNase R reaching nearly 100% depolymerization in 2 hours.
  • the remaining nucleases did not produce detectable depolymerization in this assay.
  • Subsequent analyses by LC-MS revealed NMP liberation in samples treated with RNase R and Nuclease PI, but not Benzonase or RNase A ⁇ Figure 5B). These results suggest that RNase R and Nuclease PI may be suitable for depolymerizing lysate RNA into 5'-NMPs.
  • RNase R was chosen for further study for several reasons, including its lack of DNAse activity, its ability to degrade dsRNA and structured RNA, and its processive 3 ' ⁇ 5' exonuclease activity.
  • RNase R was then tested for its ability to depolymerize endogenous RNA in bacterial lysates.
  • purified RNase R 0.5 mg/mL final concentration
  • lysates 50% final concentration
  • free nucleotides were quantified by UPLC.
  • Figure 6 A representative experiment is shown in Figure 6. Adding purified RNase R to lysates resulted in the rapid release of 5'-NMPs from lysate RNA, with maximal NMP liberation after 5-10 minutes. After this initial period of rapid depolymerization, NMP concentrations stabilized, then began to slowly decline. Endogenous RNase activity also resulted in 5'-NMP liberation, albeit at much lower rates.
  • RNase R addition did not increase the rate of 2' or 3' NMP liberation from RNA, consistent with its known mechanism of action. Across multiple independent experiments, addition of RNase R to lysates resulted in the conversion of 68% of lysate RNA to 5'-NMPs in 5-10 minutes at rates in excess of 200 mM/hr.
  • NMP pool is progressively phosphorylated to NTPs before polymerization into dsRNA.
  • Deleterious enzymatic activities such as NMP degradation into nucleosides and subsequent hydrolysis into sugars and bases, negatively impact dsRNA yields. Therefore, the stability of individual NMPs was assessed in lysates. Stability assessments were performed by adding isotopically-labeled "heavy" NMPs (hAMP, hCMP, hUMP, and hGMP) to lysates, and quantifying abundance over time using LC-MS (Figured 9A-9D, solid lines). In contrast to hAMP, which is relatively stable, hCMP, hUMP, and hGMP are actively degraded by the lysate, with approximate half-lives (t 1/2 ) of 1 hour, 30 minutes, and 20 minutes, respectively.
  • lysates were pre-incubated at elevated temperature for 15 minutes. Then, the temperature was lowered to 37°C and transcription reactants (including exogenous NTPs, DNA template, and purified T7 RNA polymerase) were added. As shown in Figure 11B, transcription reactions in lysates that were heat inactivated at >70°C yielded RNA products qualitatively similar to the positive control reaction (performed in buffer). No RNA product was apparent in the reaction performed at 60°C.
  • thermostable kinases [00178] Taken together, these results suggest that a 70°C incubation for 15 minutes is sufficient to stabilize NMPs, NTPs, and dsRNA in cell-free reactions. Selection and evaluation of thermostable kinases
  • kinase activities are required to sequentially phosphorylate 5'- MPs liberated from RNA to NTPs that can be polymerized to form dsRNA.
  • These kinases which use high-energy phosphate groups from ATP to phosphorylate MPs and DPs, must be sufficiently thermostable to remain active following high- temperature incubation, as well as sufficiently active to produce NTPs at high rates (21 mM/hr NTPs for 1 g dsRNA/L/hr).
  • Enzymes from thermophilic organisms were chosen for evaluation (Table 9) based on literature reports of successful expression in E. coli and biochemical characterization of the recombinant enzymes.
  • thermostable kinases tested for each class of activity.
  • enzymes were cloned into an E. coli protein expression vector with an N-terminal hexahistidine tag, overexpressed, and purified using immobilized metal affinity
  • T. thermophilus CMP kinases encoded by the cmk gene
  • T. thermophilus enzyme yielded soluble protein under the tested conditions.
  • T. thermophilus CMP kinase exhibited activity largely independent of temperature, although enzyme activity decreased slightly at temperatures above 60°C ⁇ Figure 14). Based on these results, expression of TthCmk in high- density lysates at 0.02% of total protein would yield CMP kinase activities of 30-45 mM/hr (depending on temperature, Table 12), well in excess of the 5.25 mM/hr target. Therefore, TthCmk was chosen for further evaluation.
  • Table 12 shows predicted Cmk reaction rates in high-density (90 g dcw/L) lysates, assuming TthCmk constitutes 0.02% of total lysate protein.
  • TmGmk Based on the measured specific activities of TmGmk, expression in a high- density cell lysate at 0.1% of total protein would yield an expected rate in excess of 60 mM/hr at 70°C in the presence of excess ATP, compared to a target rate of 5.25 mM/hr (Table 13). Therefore, TmGmk was chosen for further evaluation. Table 13 shows predicted Gmk reaction rates in high-density (90 g dcw/L) lysates, assuming TmGmk constitutes 0.1% of total lysate protein.
  • NDP kinase phosphorylates ADP, CDP, UDP, and GDP.
  • enzymes from the thermophiles T. thermophilus and A. aeolicus were cloned and expressed in E. coli. While the T. thermophilus enzyme was insoluble under the tested conditions, expression of the A. aeolicus enzyme yielded soluble protein.
  • Activity measurements in a luciferase assay using ATP and GDP as substrates revealed that AaNdk is highly active across a broad range of temperatures, with a temperature optimum of 50°C ⁇ Figure 16).
  • Ppk Polyphosphate kinase reversibly transfers high-energy phosphate groups between polymeric phosphate chains and adenosine nucleotides.
  • Ppk enzymes belonging to the Type I family of polyphosphate kinases, were evaluated for activity, including enzymes from E. coli as well as the thermophilic organisms T. thermophilus and Thermosynechococcus elongatus. These enzymes were selected for testing as they belonged to the well-characterized Type I family or had previously been shown to be active.
  • kinases were evaluated in a multi-enzyme system for their ability to convert NMPs to NTPs.
  • equal volumes of lysates expressing individual kinases (5) were combined, heat-inactivated, then assayed for ATP-dependent production of NTPs from an equimolar mix of NMPs by LC-MS.
  • overall NTP production rates exceeded 24 mM/hr for UTP, CTP, and GTP, suggesting that a simple mixture of lysates, without any optimization of reaction conditions, could provide NTPs at sufficient rates to support synthesis of 1 g/L/hr dsRNA, in the presence of adequate ATP.
  • RNA polymerase After depolymerization of RNA into NMPs and phosphorylation of NMPs to their corresponding NTPs, a RNA polymerase is required to convert NTPs into the dsRNA product.
  • RNA polymerase from the bacteriophage T7 is an attractive enzyme for use in a recombinant system for several reasons.
  • T7 RNA polymerase includes a single subunit (unlike many RNA polymerases from Bacteria and Eukarya) and has been extensively characterized by biochemical and molecular biology studies. Additionally, multiple T7 RNA polymerase mutants have been described that confer improved thermostability (see Table 18).
  • RNA polymerases were evaluated using duplex DNA template (e.g. Figure 3B) and a 37°C reaction temperature.
  • the production of RNA was quantified over time using the Quant-iT RNA Kit (Broad Range) (Thermo Fisher Scientific). Under conditions recommended by each manufacturer, the ThermoT7 and MegaScript enzymes were highly active, while the NEB enzyme displayed significantly lower activity (Figure 18).
  • RNA synthesis rates exceeded 11 g/L/hr at 50°C with duplex DNA template and ThermoT7 polymerase ( Figure 19). ThermoT7 was then selected for further characterization.
  • RNA polymerization was quantified in heat-inactivated high-density lysates (68% final lysate concentration) with and without clarification to remove precipitated proteins after heat inactivation (Figure 20).
  • RNA synthesis rates were significantly higher in matrix than in buffer, with the highest rates occurring in heat-inactivated matrix that had been clarified by centrifugation. In unclarified reactions, overall RNA synthesis rates (over a 2-hour reaction) were in excess of 2 g/L/hr with 1.4% of total protein in the assay as ThermoT7 polymerase.
  • thermostability of ThermoT7 was tested at higher temperatures to evaluate compatibility with the heat inactivation conditions established earlier in this program.
  • ThermoT7 enzyme was pre-incubated at elevated temperature (50- 70°C) for varying lengths of time (0-15 minutes), and the remaining activity quantified at 37°C.
  • incubation at 50°C is well-tolerated by the enzyme, but higher temperatures lead to rapid irreversible inactivation of polymerase activity. Therefore, in these experiments, this particular Thermo T7 was not compatible with heat-inactivation at 50-70°C.
  • purified ThermoT7 enzyme may be added to cell-free reactions following a thermal inactivation, or an alternate T7 RNA polymerase mutant may be used, having sufficient half-life at 70°C, for example.
  • AMP nucleotide monophosphates
  • CMP nucleotide monophosphates
  • UMP nucleotide monophosphates
  • GMP mass spectrometry
  • Samples were separated using an Agilent 1100 series HPLC equipped with a ZIC-cHILIC column (2.1 x 20 mm, 3 ⁇ i.d.) (Merck) at room temperature with a flow rate of 0.5 mL/min and a 2 ⁇ _, injection volume.
  • Mobile phases consisted of 20 mM ammonium acetate (A) and 20 mM ammonium acetate in 90% acetonitrile (B).
  • the separation method consisted of a gradient from 15-50% (B) for 3.5 minutes, followed by 50% (B) for 1.5 minutes, then 15% B for 3 minutes. Quantification was performed on an ABSciex API 3200 mass spectrometer using electrospray ionization (capillary voltage: -3000V, temperature: 600°C, desolvation gas: 20 psi) in multiple reaction monitoring (MRM) mode. Analysis of nucleotide monophosphate, diphosphate, and triphosphate species (NMPs, NDPs, and NTPs) used the method described above with the following separation gradient: 15% B to 50% B for 3.5 minutes, followed by 50%) B for 2.5 min, then 15%> B for 4 minutes.
  • NMPs, NDPs, and NTPs nucleotide monophosphate, diphosphate, and triphosphate species
  • Peak areas were compared to standard curves consisting of purified compounds (Sigma-Aldrich).
  • standard curves were prepared in lysate backgrounds that had been acid-quenched, clarified, pH-neutralized, and filtered as in the sample preparation steps described below.
  • the separation method consisted of 1% B for 2.8 minutes, followed by a gradient from l%-30% B for 2.2 minutes, followed by 100% B for 7 minutes, then 1% B for 3 minutes. Quantification was performed using an ACQUIT Y UPLC PDA (Waters) at 260, 254, and 210 nm. Peak areas were compared to standard curves consisting of purified compounds
  • Pellets were resuspended in 1 mL of a 2M solution of lithium chloride (Sigma-Aldrich) in 35% ethanol. The suspensions were incubated at room temperature for 5 minutes, then clarified by centrifugation at 15,000 x g for 6 minutes at 4°C and the supernatants removed. Pellets were then resuspended in 1 mL of a 2M solution of lithium chloride in water, and incubated at room temperature for 2 minutes before clarification at 15,000 x g for 6 minutes. Supernatants were then removed and the remaining pellets washed by resuspending in 70% ethanol and centrifuging at maximum speed (21,000 x g) for 5 minutes at 4°C.
  • a 2M solution of lithium chloride Sigma-Aldrich
  • RNA solutions were clarified by centrifugation (maximum speed for 5 minutes at 4°C) and supernatants containing soluble RNA were transferred to sterile RNase-free tubes and stored at -20°C.
  • 2X assay buffer 100 mM potassium phosphate pH 7.4, 10 mM magnesium chloride, 1 mM zinc chloride
  • Reactions were incubated at 37°C and periodically sampled by transferring 20 ⁇ _, to acid quench solution (180 ⁇ _, of 0.2M sulfuric acid) on ice. After completion of the time course, quenched samples were clarified by centrifugation at 3,000 x g for 5 minutes at 4°C. 170 ⁇ _, of supernatant from each sample was then transferred to a UV-transparent 96-well half area plate (Corning) and acid- soluble nucleotides were quantified by absorbance at 260 nm using a microplate reader and an extinction coefficient of 10665 M "1 cm "1 , estimated by averaging individual extinction coefficients for each mononucleotide.
  • Lysates were then clarified by centrifugation at 16,000 x g for 1 hour at 4°C. Proteins were purified by immobilized metal affinity chromatography using His GraviTrap columns (GE Healthcare) or HisTrap HP columns connected to an AKTAPrime Plus FPLC system (GE Healthcare). For both purification methods, columns were
  • Equilibration/Wash and Elution buffers used 50 mM Tris-HCl pH 7.5 instead of phosphate buffer. Elution fractions were analyzed by SDS-PAGE and protein content quantified by BCA (Thermo Fisher Scientific). Fractions were then combined and buffer exchanged by dialysis into 1000 volumes 2X Storage Buffer.
  • 2X Storage Buffer consisted of 2X PBS supplemented with an additional 500 mM NaCl.
  • 2X Storage Buffer consisted of 2X PBS.
  • 2X Storage Buffer consisted of 100 mM Tris-HCl pH 7.0 with 100 mM NaCl. After dialysis, proteins were mixed with an equal volume of 100% glycerol (50% final concentration) and stored at -20°C.
  • E. coli strains GL16-170 (BL2 ⁇ (DE3).t526pgi.Aedd.AtktB.AtolC_wt-7-
  • GL16-170 lysate protein content 34.5 mg/mL
  • RNase R solution (1 mg/mL in 300 mM potassium phosphate buffer pH 7.4, 200 mM KC1, 2 mM MgCl 2 ) were pre-equilibrated at 2°C before initiating the reaction.
  • time t 0, 50 ⁇ _, of E. coli lysate and 50 ⁇ _, RNase R solution were mixed and the reaction initiated by transferring to a preheated 37°C block.
  • Reactions including deoxycholate were assembled as described above, except that lysates were premixed with 0.2 volumes of 5X sodium deoxycholate solutions in water and incubated at 2°C for 15 minutes before initiation.
  • reaction After initiation, reactions were incubated at 37°C and periodically sampled by transferring 10 ⁇ . to acid quench solution (90 ⁇ . of 0.2M sulfuric acid) on ice. After completion of the time course, quenched samples were clarified by centrifugation at 3,200 x g for 10 minutes at 2°C. Depolymerization was first quantified by absorbance of acid-soluble nucleotides: 10 ⁇ . of quenched and clarified reactions was added to 160 ⁇ . of 0.2M sulfuric acid in a UV-transparent 96-well half area plate (Corning). Acid-soluble nucleotides were quantified by absorbance at 260 nm using a microplate reader (see above).
  • Depolymerization was also quantified by UPLC analysis of 5', 2', and 3' NMPs: 30 ⁇ . of each acid-quenched sample was pH-neutralized by adding 10 ⁇ . of 1M KOH, then passed through a 0.2 ⁇ filter before UPLC analysis. The total nucleotide pool (i.e. 100% depolymerization) was determined by alkaline hydrolysis of lysate RNA: 50 ⁇ , lysate was combined with 150 ⁇ L of 0.2M potassium hydroxide, then heated to 99°C for 20 minutes. Alkaline-hydrolyzed samples were then quenched and analyzed as described above.
  • Lysates were prepared from frozen biomass as described above (Protein concentrations: 36.6 mg/mL for GL16-170 biomass with empty pETDuet-1; 53.2 mg/mL for GL16-170 with pETDuet-1 carrying cloned RNase R).
  • Depolymerization in dilute lysates was assessed as described above with 50% final lysate concentration in the reaction.
  • Depolymerization in concentrated lysates was assessed by pre- incubating 9 volumes lysate with 1 volume 10X EDTA solution for 5 minutes at 2°C.
  • Lysates and NMPs were equilibrated to 37°C for 10 minutes before reactions were initiated.
  • 90 ⁇ _ lysate solution was added to 10 ⁇ _, NMP solution, and the reactions mixed well. Reactions were monitored by sampling at the indicated time points. During sampling, 12 ⁇ _, of reaction mixture were transferred to 108 ⁇ _, of 0.2M sulfuric acid on ice. Quenched reactions were then clarified by centrifugation, pH- neutralized, and filtered for LC-MS analysis as described above.
  • GL14-322 lysate was aliquoted into 5 microcentrifuge tubes on ice, then transferred to a heat block equilibrated at the desired heat inactivation temperature. At the indicated times, tubes were cooled on ice, then clarified by centrifugation (21,000 x g for 5 minutes) and the supernatants harvested. Supernatants from heat-inactivated lysates, along with an equimolar mixture of NTPs (Sigma- Aldrich, 25 mM each) were equilibrated at 37°C for 10 minutes.
  • Nucleotide kinases were assayed at varying temperatures (37°C, 50°C, 60°C, 70°C, and 80°C) in a buffer consisting of 50 mM Tris-HCl pH 7.0, 4 mM MgS0 4 , 4 mM ATP, and 4 mM of the corresponding NMP or NDP.
  • Reaction buffer 1.2X concentrate
  • enzyme solution 0.5 mg/mL
  • reaction mixture 15 ⁇ _, of reaction mixture were transferred to 135 ⁇ _, of 0.2M sulfuric acid on ice. After completion of the reaction, samples were pH-neutralized with 1M KOH as described above, then diluted 1 : 10 in ice-cold water. ATP was quantified in each sample using the ATP Bioluminescent Assay Kit (Sigma-Aldrich), following kit instructions.
  • lysates were aliquoted into individual reaction tubes, then heat-inactivated by incubating at 70°C for 15 minutes.
  • Reaction buffer (2X concentrate) and heat-inactivated lysates were pre-equilibrated at reaction temperature, and the reactions initiated by combining equal volumes of lysate and reaction buffer. Reactions were sampled by quenching individual reaction tubes with 9 volumes acid quench solution, then analyzed as described above.
  • lysates individually expressing each kinase were mixed in a 1 : 1 ratio, divided into 10 ⁇ , aliquots, then heat-inactivated as described above.
  • Kinase activity was analyzed in a buffer consisting of 50 mM Tris-HCl pH 7.0, 16 mM MgS0 4 , 2 mM each nucleotide monophosphate (AMP, CMP, UMP, and GMP), and 16 mM ATP.
  • Reaction buffer (2X concentrate) pre-equilibrated at reaction temperature was combined with an equal volume of lysate to initiate the reaction. Reactions were performed at 70°C and sampled by quenching individual reaction tubes with 9 volumes acid quench solution, then analyzed as described above.
  • reaction buffer consisting of 50 mM Tris-HCl pH 7.0, 4 mM MgS0 4 , 25 mM (NH 4 ) 2 S0 4 , 1 mM ADP, and 1 mM sodium hexametaphosphate.
  • Reaction buffer (1.2X concentrate) and enzyme solution (0.25 mg/mL) were pre-equilibrated at reaction
  • lysates were aliquoted into individual reaction tubes, then heat-inactivated by incubating at 70°C for 15 minutes.
  • Reaction buffer (2X concentrate) and heat-inactivated lysates were pre-equilibrated at reaction temperature, and the reactions initiated by combining equal volumes of lysate and reaction buffer. Reactions were sampled by quenching individual reaction tubes with 9 volumes acid quench solution, then analyzed as described above.
  • Reaction rates in lysates were calculated by subtracting signal from a control lysate (without overexpressed polyphosphate kinase) under the same reaction conditions.
  • Duplex DNA template was prepared by PCR amplification of synthetic gBlock DNA (Integrated DNA Technologies). Reactions were purified and concentrated by isopropanol precipitation.
  • RNA polymerases were compared using conditions recommended by each manufacturer. Each 50 ⁇ _, reaction consisted of 10X reaction buffer (supplied by the manufacturer), NTPs, DNA template (0.5 ⁇ g), and enzyme. For the NEB T7 RNA polymerase, reactions included 0.5 mM each NTP, 5 mM DTT, and 100 U enzyme. Reactions with ThermoT7 polymerase were identical, except that DTT was omitted.
  • Reactions with MegaScript T7 included 7.5 mM each NTP and 5 ⁇ _, enzyme mix. Enzyme concentrations were determined by BCA assay (Thermo Fisher). Reactions were monitored by sampling at the indicated time points. During sampling, 10 ⁇ _, of reaction mixture were transferred to 90 ⁇ , of RNA quench solution (10 mM Tris-HCl pH 8.0, 5 mM EDTA) and stored on ice. RNA samples in quench solution were quantified using the Quant-iT RNA Broad Range Assay Kit (Thermo Fisher), following kit instructions. Serial dilutions of purified dsRNA, prepared using the MegaScript Kit and purified following kit instructions, were used to construct standard curves for quantitation. Reactions were qualitatively analyzed by agarose gel electrophoresis.
  • GL14-322 lysates were heat-inactivated and clarified by centrifugation as described previously.
  • Each 20 ⁇ _, reaction consisted of clarified lysate (7 ⁇ .), 10X cofactor solution (300 mM MgCl 2 , 20 mM spermidine), NTPs (7.5 mM each, prepared from pH- neutralized stock solutions), DNA template (0.6 ⁇ g), and enzyme (1 ⁇ ).
  • Reactions were incubated for 1 hour at 37°C or 50°C, then quenched by adding 9 volumes RNA quench solution. Quenched reactions were further diluted 10-fold in quench solution (final dilution: 100-fold). Diluted reactions were then quantified using the Quant-iT kit (see above).
  • RNA produced by the reaction was calculated by subtracting RNA quantified in a control reaction (omitting RNA polymerase).
  • RNA polymerase assays in high-density lysates were performed as described above, with the following modifications.
  • Each 100 ⁇ . reaction consisted of lysate (67.5 ⁇ .), 10X cofactor solution (300 mM MgCl 2 , 20 mM spermidine), NTPs (7.5 mM each, prepared from pH-neutralized stock solutions), DNA template (3 ⁇ g), and enzyme (10 ⁇ .).
  • GL14-322 lysates (67.5 ⁇ .) were aliquoted into individual reaction tubes, then heat-inactivated as described previously.
  • Thermostable PPK2 enzymes were codon-optimized for expression in E. coli, synthesized, and cloned into pETDuet-1 (Novagen). Plasmids were then transformed into GL16-170. To generate the Control strain, empty pETDuet-1 plasmid was transformed into GL16-170. After overnight preculture in 5 mL Lysogeny Broth (LB), strains were cultivated in 1L LB at 37°C until cell densities reached an OD 60 o of approximately 0.5. Cultures were then briefly chilled on ice, and PPK2 expression was induced by adding isopropyl thiogalactopyranoside (IPTG) to a final concentration of 0.25 mM.
  • IPTG isopropyl thiogalactopyranoside
  • Thawed lysates expressing PPK2 enzymes were first diluted 1 : 100 into lysates prepared from the Control strain, except for the D. geothermalis PPK2 lysate, which was diluted 1 : 10.
  • Pre- chilled solutions of manganese chloride (MnCl 2 ) and sodium hexametaphosphate (HMP) were added to final concentrations of 10 mM and 1 mM, respectively. Lysates were then heat-inactivated by incubation in a pre-heated 70°C thermocycler for 15 minutes.
  • Reactions were then initiated by mixing heat-inactivated lysates with an equal volume of 2X Reaction Buffer, consisting of 10 mM MnCl 2 , 2 mM adenosine diphosphate (ADP) or adenosine monophosphate (AMP), and 9 mM HMP. Reactions were incubated at 70°C, and time points were taken by removing an aliquot of reaction mixture and diluting with 9 parts Quench Solution (200 mM H 2 S0 4 ) on ice. The initial timepoint (to) was taken by directly mixing lysate with Quench Solution, storing the quenched lysate on ice for 15 minutes, then adding 2X reaction buffer.
  • 2X Reaction Buffer consisting of 10 mM MnCl 2 , 2 mM adenosine diphosphate (ADP) or adenosine monophosphate (AMP), and 9 mM HMP. Reactions were incubated at 70°C, and time points
  • quenched timepoint solutions were clarified by centrifugation at 3,200 x g for 10 minutes.
  • Supernatants from the quenched reactions were then pH neutralized by mixing 3 parts quenched reaction solution with 1 part Neutralization Solution (1M KOH).
  • Quenched and neutralized samples were then diluted 1 : 10 with water before quantitation using the Adenosine 5 '-triphosphate (ATP)
  • Bioluminescent Assay Kit (Sigma-Aldrich cat #: FLAA), following kit instructions. Initial reaction rates were calculated based on the accumulation of ATP in PPK2-containing reactions, subtracting ATP concentrations from the Control lysate.
  • Table 17 Summary of expression and rate data for thermostable Class III PPK2 enzymes in lysates. Roseiflexus sp. RS-1 ++ 680 470
  • Thermostable C. aerophila PPK2 was then used to supply ATP for cell-free production of dsRNA from NMPs, ADP, and HMP.
  • Cell-free dsRNA synthesis reactions were performed with a mixture of six E. coli lysates individually overexpressing the kinases detailed in Table 18.
  • lysates detailed in Table 18 were combined in equal volumes on ice. Pre- chilled solutions of manganese chloride (MnCl 2 ), magnesium sulfate (MgS0 4 ), and sodium hexametaphosphate (HMP) were added to final concentrations of 0 - 2.5 mM, 30 mM, and 1 mM, respectively. The lysate mixture was then then heat-inactivated by incubation in a preheated 70°C thermocycler for 15 minutes.
  • MnCl 2 manganese chloride
  • MgS0 4 magnesium sulfate
  • HMP sodium hexametaphosphate
  • heat-inactivated lysates were combined with the following components: an equimolar mixture of nucleotide monophosphates (adenosine 5 '-monophosphate, cytidine 5 '-monophosphate, uridine 5'- monophosphate, and guanosine 5 '-monophosphate, 2 mM each), 50 mM Tris pH 7.0, 30 mM MgS0 4 , 0 - 2.5 mM MnCl 2; 1 mM adenosine 5 '-diphosphate, 2 mM spermidine, 1.5 ⁇ g plasmid DNA template, and 3 ⁇ g thermostable T7 RNA polymerase (S430P, F849I, F880Y) in a total volume of 20 ⁇ ⁇ .
  • nucleotide monophosphates adenosine 5 '-monophosphate, cytidine 5 '-monophosphate, uridine 5
  • dsRNA was synthesized from an equimolar mixture of 2 mM NTPs (with lysates, ADP, and HMP omitted).
  • dsRNA was synthesized from an equimolar mixture of 2 mM NMPs (with PPK2-expressing lysate, ADP, and HMP omitted, but including 8 mM ATP as an energy source).
  • duplicate reactions were performed omitting polymerase. All reactions were incubated at 50°C for 2 hours, then terminated by the addition of 9 volumes TE+ buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA). Samples were mixed with an equal volume of 2X RNA Loading Dye (New England Biolabs) and heated to 70°C for 10 minutes, followed by agarose/TAE gel electrophoresis.
  • the desired dsRNA product was produced in buffer using NTPs (left lanes), in nucleotide kinase-expressing lysates from NMPs and ATP (middle lanes), and in nucleotide kinase and polyphosphate kinase-expressing lysates from NMPs and HMP (right lanes).
  • Manganese chloride was not required in any reaction, demonstrating that the C. aerophila enzyme can utilize Mg 2+ as a cofactor as well as Mn 2+ . Therefore, Mn 2+ is not required a priori for cell-free reactions containing C. aerophila PPK2.
  • the desired dsRNA product was produced in buffer using NTPs (left lanes), in nucleotide kinase-expressing lysates from NMPs and ATP (middle lanes), and in nucleotide kinase and polyphosphate kinase-expressing lysates from NMPs and HMP (right lanes).
  • dsRNA production from NMPs and HMPs did not require exogenous ADP or T. thermophilus AMP kinase. Therefore, C. aerophila PPK2 can be used as part of a 5-kinase system to produce dsRNA from NMPs and HMP in cell-free reactions.
  • the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features.
  • citrinum PI nuclease at 2.8- A resolution EMBO J. 10: 1607-1618(1991)
  • thermophilus Adk T. thermophilus Adk

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Abstract

La présente invention concerne dans certains aspects, des méthodes et des compositions pour la production acellulaire d'acide ribonucléique.
PCT/US2017/026285 2016-04-06 2017-04-06 Production acellulaire d'acide ribonucléique WO2017176963A1 (fr)

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BR112018070506A BR112018070506A2 (pt) 2016-04-06 2017-04-06 produção isenta de células de ácido ribonucleico
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CR20180525A CR20180525A (es) 2016-04-06 2017-04-06 Producción de ácido ribonucleico libre de células
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EP17722192.6A EP3440215A1 (fr) 2016-04-06 2017-04-06 Production acellulaire d'acide ribonucléique
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